1
INTRODUCTION
The current investigations concern a study of the ADP-ribosylation reaction
catalyzed by the exotoxin A, (ETA), produced by Pseudomonas aeruginosa. The
phenomenon of ADP-ribosylation is widespread in the biological kingdom. While it
provides, in general, a mechanism by which living systems can regulate their metabolic
activities, it can also have detrimental effects when used by a pathogenic organism as a
virulence determinant.
As the name indicates, ADP-ribosylation involves covalent attachment of an
ADP-ribosyl moiety, ADPR, to a specific amino acid residue in the target protein. The
source of ADPR is NAD+ and, hence, one of the substrates in the reaction catalyzed by a
family of enzymes referred to as ADP-ribosyl transferases, ADPRTs. Investigations
during the past couple of decades have given new insights into the novel contributions of
this pyridine dinucleotide to a variety of intracellular metabolic functions. Hence, a brief
review of the processes which are dependent on the participation of NAD+ (or its
derivative) are appropriate prior to focusing attention on the specific topic of the current
investigations.
NAD+ functions as a hydride donor or acceptor in numerous metabolic processes
and its role in energy transduction has been recognized for several decades (Warburg et.
al., 1935). The ability of NAD+ to serve in other capacities was first noted when it was
found that it could serve as a substrate for effecting covalent modification of proteins
(Chambon et. al., 1963; Fujimura et. al., 1967; Nishizuka et. al., 1967). The initial
observation that NAD+ serves as a substrate in the diphtheria toxin (DT) catalyzed ADPribosylation of amino acyl transferase II (Honju et. al., 1968) was followed by the
2
identification of other bacterial toxins capable of displaying similar catalytic activity
(Sekine et. al., 1989; Vandekerckhove et. al., 1987; Collier 1975). A number of
eukaryotic ADPRTs have been recorded, the highly investigated of this type being poly
(ADP-ribosyl) polymerase (PARP1).
1.1
ADP-Ribosylation
The process of ADP-ribosylation can occur either as a non-enzymatic or an
enzyme catalyzed event. In the former case, the ADP-ribosyl moiety of NAD+ becomes
attached, nonenzymatically, through Schiff base formation to a reactive functional group
such as a cysteine(s) or a lysine(s) present in the protein (Cervantes-Laurean et. al., 1993
& 1996). However, the significance of such reactions to signaling pathways in vivo
remains obscure. In the enzyme-mediated process, the ADPR moiety of NAD+ is
transferred to specific proteins as shown in Figure 1. This type of protein glycation
serves as an important means of regulating the intracellular activity of enzymes.
1.2
Mono ADP-Ribosylation
The mono ADP-ribosyl transferases (mADPRTs) have been classified on the
basis of the specificity of the amino acid residue in the target protein, that serves as the
acceptor of the ADPR moiety. The amino acid residues serving as sites of ADPribosylation can be cysteine (as in pertussis toxin), arginine (cholera toxin) or
diphthamide (DT & ETA). The majority of eukaryotic ADPRTs seem to have an
arginine as the acceptor site.
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4
The mechanism involved in the mADPRT catalyzed reaction involves a direct
transfer of the ADPR moiety from -NAD+ to the acceptor site. The  isomer of NAD+
is not a substrate for ADPRTs.
As mentioned above, bacterial toxins effect ADP-ribosylation of specific
protein(s) of host cells often resulting in lethal effects (Moss & Vaughan 1988).
Interestingly, most of these toxins use G-proteins as their target. Thus, for example, CT
catalyzes ADP-ribosylation of the -subunit of Gs (Cassel & Pfeuffer 1978), with
arginine as the acceptor amino acid residue. Such modification results in the loss of
GTPase activity with concomitant activation of adenylyl cyclase (Moss & Vaughan
1977). The mono ADP-ribosylation of G-proteins is shown in Figure 2.
As noted above, eukaryotic mADPRTs modify an arginine residue of their target
proteins. Since they are either glycosyl phosphatidyl inositol anchored or secreted, their
action appears to be in the regulation of extracellular rather than of intracellular events
(Okazaki & Moss 1998).
Although arginine specific ADP-ribosylation prevails in the case of these
enzymes, other amino acid(s) can serve as acceptors. Indeed the regulation of glutamate
dehydrogenase activity in mitochondria by ADP-ribosylation of one of its cysteine
residues has been reported recently (Herrero-Yraola et. al., 2001). The possibility of an
ADP-ribosylation cycle has been raised by the report on the isolation and characterization
of ADP-ribosyl arginine hydrolase (Moss et. al., 1985).
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6
1.3
Poly (ADP-ribosyl)ation
The phenomenon of poly (ADP-ribosyl)ation of proteins appears to be involved in
the stimulation of DNA repair (Durkacz et. al., 1980, Satoh & Linahl 1992) and other
fundamental processes related to the maintenance of the functional integrity of the
genome (Lindahl et. al., 1995; Oei et. al., 1997; Jeggo 1998).
The level of poly (ADP-ribosyl)ation in cells appears to be the most important
determinant for the maintenance of NAD+ levels (Hillyard et. al., 1981; Carson et. al.,
1986, Schraufstatter et. al., 1986). The catabolism of NAD+ in vivo appears to proceed
via poly (ADP-ribosyl) ation reaction. DNA damaging agents effect depletion of
intracellular NAD+ levels, which results in the depletion of ATP. Furthermore, such
depletion of NAD+ also prevents generation of ATP linked to the dehydrogenation of
glyceraldehyde-3-phosphate in the glycolytic pathway (Jacobson et. al., 1979; Bernofsky
1980; Goodwin et. al., 1978).
The poly (ADP-ribosyl) ation process requires the generation of a homo polymer
of ADP-ribose, PADPR, the synthesis of which is catalyzed by PARP-1, an enzyme of
molecular weight ~113 kDa (Lindahl et al., 1995). This enzyme has three distinct
catalytic activities required for performing poly (ADP-ribosyl) ation in living cells.
These activities are: (i) initiation of mono ADP-ribosylation on a protein target; (ii)
elongation of the polymer; and (iii) branching of the polymer.
The enzymatic degradation of PADPR requires three distinct enzymatic activities
performed by two different proteins, PADPR glycohydrolase (PARG) and ADP-ribosylprotein lyase. The events involved in PADPR metabolism are shown in Figure 3. More
than 30 distinct PARPs have been identified. The above mentioned PARP-1 is referred
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8
to as type I PARP. The others fall into the type II and type III categories (Babiychuk et.
al., 1998; Smith et. al., 1998; Faraone-Mennella et. al., 1998). These proteins are
involved in the metabolism of nucleic acids and in the maintenance of chromatin
integrity.
The main acceptor of PADPR is PARP-1 itself as it catalyzes its own
modification to complete its shutting of DNA structural breaks. PARP-1 is a
multifunctional protein, comprising three domains, the DNA binding domain (DBD), the
automodificaiton domain and the catalytic domain (Figure 4).
DNA is the essential element in the regulation of poly (ADP-ribosyl) ation
reactions. Double stranded breaks in DNA stimulate the activity of PARP-1 500 fold
(Alvarez-Gonzalez & Althaus 1989; Simonin et. al., 1993). The automodification that
follows this activation regulates PARP-1 activity. The sequences of events
accompanying the automodification of PARP-1 are shown in Figure 5. The catalytic
domain of PARP-1 has structural features similar to that of mADPRTs. The role of
PARPs in regulation of nuclear function has been a topic of recent review (D’Amours et.
al., 1999).
1.4
Cyclic ADP-ribose (cADPR)
The observation that the addition of NAD+ to homogenates of sea urchin egg
caused a release of Ca2+ ions provided an impetus to studies which led to the
identification of cyclic ADPR as a potent intracellular calcium mobilizing agent (Clapper
et. al., 1987; Lee 1997). The structure is unique in that cyclization occurs between the
anomeric carbon of the terminal ribose and the N1 nitrogen of the adenine ring. The
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10
11
reaction is catalyzed by NAD+ glycohyrolase, which normally gives rise to ADPR and
nicotinamide. In view of the presence of both hydrolytic and cyclization activities in
glycohydrolases, these enzymes are referred to as ADP-ribosyl cyclases. NAD+
glycohydrolases studied to date show both types of activities, the exception being that
from Neurospora crassa which is devoid of cyclase activity (Graeff et. al., 1994). The
reaction mechanism of NAD+ glycohdyrolase/cyclase is shown in Figure 6. The presence
of ADPR cyclase in prokaryotes suggests that signaling via cADPR is a common wellpreserved mechanism (Karasawa et. al., 1995).
From the account presented above, it is evident that NAD+ plays a pivotal role in
the regulation of the intracellular metabolic functions. The current dissertation concerns
a study of the mono ADP-ribosylation reaction mediated by the exotoxin A produced by
Pseudomonas aeruginosa. This process provides the organism with a means for
debilitating the host’s protein synthesis machinery and thus facilitates its unimpeded
proliferation. A brief review of the information currently available on both ETA and its
protein target, elongation factor 2 (EF-2) is provided below.
1.5
Exotoxin A
Exotoxin A, first identified by Liu and coworkers as a lethal virulence factor
produced by P. aeruginosa (Liu et. al., 1966), turns out to be one of the most potent
toxins with a lethal dose of 0.2 g/kg when administered to mice intraperitoneally
(Iglewski et. al., 1979). ETA belongs to a family of bacterial toxins that catalyze ADPribosylation of specific eukaryotic proteins. ETA, like DT, catalyzes the ADP-
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13
ribosylation of the diphthamide residue of EF-2 resulting in the cessation of protein
synthesis in the host. In general, the ADP-ribosylating toxins are composed of a catalytic
and a translocation moiety. The catalytic moiety once in the cytoplasm of the host cell, is
responsible for ADP-ribosylating an acceptor protein, which ultimately results in cell
death or malfunction.
ETA is produced as an inactive precursor and requires processing prior to
emergence of its ADPRT activity. In vivo, these alterations include proteolytic cleavage
of domains I and II from the catalytic domain of the protein, followed by the reduction of
a disulfide bridge in the heterodimeric product. The conditions for achieving the in vitro
activation of ETA have been documented (Leppla et. al., 1978). The nucleotide sequence
of tox A, the gene encoding for ETA has been determined (Gray et. al., 1984). The
determined amino acid sequence of ETA and its various functional domains are shown in
Figure 7.
1.6
Structure of Exotoxin A
The three dimensional structures of the proenzyme form of ETA and of its C-
domain have been determined by X-ray crystallography (Allured et. al., 1986, Li et. al.,
1995). The crystal structures of C-terminus domain of ETA in complex with
nicotinamide and ADP-ribose as well as with -TAD (Li et. al., 1996) have been
determined.
Studies based on structural elucidation of ETA and analysis of its variants
obtained by deletion mutations (Hwang et. al., 1987) have provided valuable insight into
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15
16
the organization of its domains (Figure 8). These can be classified as receptor binding
domain (I), translocation domain (II), and the catalytic domain (III). The receptor
binding domain is made of two polypeptide segments, the first, Ia, comprising amino acid
residues 1-253 and the second, Ib, containing amino acid residues 365-404. This domain
has 17 -strands (anti parallel), thirteen of which form the structural core of an elongated
-barrel. Segment Ia is essential for binding of the toxin to the target host cell (Jinno et.
al.,1988). The function of Ib remains to be determined.
Residues 253-364 of ETA constitute its translocation domain and it consists of 6
-helices with one disulfide bridge between helices A and B. This domain is responsible
for translocation of the toxin across intracellular membrane.
The catalytic domain, containing residues 453-617, is the carboxyl terminal
portion of the protein and has little structural organization relative to the other two
domains. However, two approximately orthogonal -sheets flanked by helices provide a
cleft for binding of the substrate NAD+. The NAD+ binding segment of the protein is
distinct from the traditional  motif (Rossmann et. al., 1975) typically encountered in
other proteins requiring this cofactor for their function.
The active site of ETA comprises amino acid residues needed for both the proper
interactions with the substrates as well as for its catalytic function. The nicotinamide
moiety of NAD+ appears to interact with Tyr 470 and Tyr 481 which are located in the
proximal -helix and -strand respectively, and these stacking interactions appear to
stabilize the binding of this substrate (Brandhuber et. al., 1988).
The catalytic domain of ETA, in addition to Tyr 470 and Tyr 481, accommodates
His 428, Tyr 466 and Glu 553 and the peptide segment with the amino acid sequence,
17
REDLK. The interaction of various amino acid residues present in the catalytic domain
with substrates NAD+ and EF-2 has been recorded (Tweten et. al., 1985; Douglas et. al.,
1987).
Photoaffinity labeling studies have revealed Glu 553 to be essential for catalytic
function (Carroll et. al., 1987). Its position in the active site is such as to participate in
the hydrolysis of the N-glycosidic bond of NAD+ and this view derives support from the
observation that exposure of ETA.NAD+ complex to UV irradiation results in the transfer
of the ADPR segment to the carboxyl of Glu553 (Carroll et. al., 1987).
The His 440 is located at the bottom of the binding pocket and its imidazole ring
is protruding into the cavity with rotational freedom, which facilitates its involvement in
hydrogen bonding. This residue is strictly conserved among other members of ADPribosylating toxins. Mutations of this residue have resulted in dramatic reductions in
ADPRT activity while glycohydrolase activity is unaffected (Xiang et. al., 1995). These
authors have proposed that His 440 might be involved with EF-2 binding.
His 426, located within an -helix comprising residues 421-432, is distal from the
catalytic site of ETA. Mutation of the tox A gene has implicated a catalytic role for His
426. In these studies, Iglewski and coworkers isolated a mutant strain of P. aeruginosa
that produces a nontoxic, enzymatically inactive, full-sized ETA protein called CRM 66
(Cryz et. al., 1980). However, there is no evidence for a direct catalytic role of His 426 in
ADPRT activity of ETA. Substitution of His 426 with Pro results in a reduction of the
ADPRT activity supporting the view that flexibility near this residue may be essential for
the protein’s catalytic function.
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In the absence of EF-2, a second type of enzymatic activity associated with ETA,
namely, a weak NAD+-glycohydrolase activity can be detected. In this reaction, the Nglycosidic bond is cleaved by water acting as the nucleophile. The glycohydrolase
activity associated with a catalytic fragment of ETA (PE40) has been determined (Beattie
et. al., 1996). This glycohydrolase activity of ETA serves as a convenient probe for
assessing the structural integrity of its NAD+ binding pocket of the protein.
Investigations based on (i) elucidation of three-dimensional structure, (ii)
photoaffinity labeling, and (iii) mutagenesis of ETA, have provided insight into the steps
associated with the ADP-ribosylation process. The reaction would appear to involve an
SN1 nucleophilic substitution mechanism in which diphthamide residue of EF-2 attacks
the anomeric carbon of ribose of ADPRETA complex, en-route to its becoming
modified by ADP-ribosylation. As outlined in Figure 9, ETA uses two substrates, NAD+
and EF-2, during the course of its catalytic cycle. The order of reaction, as proposed by
Galloway and coworkers, (Kessler et. al., 1992) involves the initial formation of a
complex of NAD+ and ETA. This is followed by binding of EF-2 to the binary complex,
and finally transfer of the ADP-ribose moiety of NAD+ to the diphthamide of EF-2. This
mechanism was proposed based on studies using a solid-phase immobilized binding
assay with EF-2 immobilized. It is noteworthy that in this assay the ADPRT reaction
does not take place due to immobilization of EF-2. However, similar studies by Peterson
and coworkers illustrate that the presence of NAD+ is not a prerequisite for the interaction
of ETA with EF-2 (Elazim et. al., 1998).
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20
1.7
Mode of entry of ETA
A schematic representation of the mode of entry into a target cell used by ETA is
shown in Figure 10. The interaction of ETA with mammalian cells is a complex multistep process that begins with receptor-mediated endocytosis and ends with covalent
modification of EF-2 leading to cell death. One of the key features of this process
includes the reduction and physical separation of the catalytic domain from the remainder
of the toxin. ETA enters a mammalian cell by specifically binding to a protein receptor
known as 2-macroglobulin/LDL receptor (Kounnas et. al., 1992). This receptor is
highly conserved in nature and is known to mediate the binding and uptake of
physiologic ligands in mammalian cells.
The surface-bound toxin enters the target cell through endocytosis. Endocytosis
takes place through two separate mechanisms: the first mechanism involves coated pits
and vesicles where as the second mechanism is not as well characterized. For ETA, the
major part of uptake appears to take place by endocytosis from coated pits (FitzGerald et.
al., 1980).
Once in the endosome the preenzyme form of ETA is cleaved by a membrane
associated protease similar to furin (Ogata et. al., 1990). This furin-like protease effects
the hydrolysis of the Arg 279-Gly 280 peptide bond, resulting in the generation of two
polypeptide chains linked by a disulfide bond. The translocation of the 37 KDa fragment
(II and III) is not completely understood. However, the available evidence suggests that
the toxin utilizes the retrograde transport machinery of the host cell to traverse from the
trans Golgi network to the ER. As mentioned earlier, a conserved amino acid sequence
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22
located in the C-terminal segment of ETA has been shown to be required for ETA’s
cytotoxic activity (Chaudhary et. al., 1990). This amino acid motif, which is similar to
the ER retention signal, KDEL, appears responsible for the transport of the 37-kDa
fragment by the retrograde pathway.
At the present time, the exact mechanism involved in the reduction of the
disulfide bridge in the fragment remains unclear. Such reduction can occur either in the
lumen of the ER (before translocation) or in the cytoplasm (after its translocation).
However, a protein disulfide isomerase present in ER has been shown to be capable of
catalyzing the scission of the disulfide bridge in the 37-kDa heterodimer (Mckee et. al.,
1999). The release of the polypeptide chain, capable of catalytic function, from its
heterodimeric state would appear to be accompanied by its unfolding, a feature required
for its transport to the cytoplasm. Upon its localization in the cytoplasm, the fragment is
rapidly refolded and restored to its catalytically competent conformation.
1.8
Elongation Factor 2
The eukaryotic elongation factor 2, EF-2, the specific target for ADP ribosylation
by ETA, is a protein of molecular weight approximately 100 kDa. It is a member of the
family of cellular GTPases and catalyzes the terminal step in the elongation cycle,
translocation, in protein synthesis (Hashimoto et. al., 1996). The primary structure of
EF-2 is highly conserved with  85% similarity being noted between the proteins of yeast
and human origin (Cammarano et. al., 1999). Such sequence similarity appears to extend
to the related, EF-Gs that occur in prokaryotes, mitochondria and chloroplast.
23
A unique feature of EF-2 concerns the post-translational modification of its single
histidine residue. This process involves introduction of a C3 unit (1-carboxy, 1-amino
propyl) at the C2 position of the amino acid followed by amidation of the carboxyl
function and trimethylation of the amino group. The source of carbon (C3 and C1) for
this modification is S-adenosyl methionine. Such modified histidine is referred to as
diphthamide and this residue is the site of ADP-ribosylation by ETA. It should be noted
that diphthamide is present only in EF-2 and not in EF-G.
The 3D structures of both EF-G from Thermus thermophilus as well as of its
complex with GDP have been determined (Czworkowski et. al., 1994, Aevarsson et. al.,
1994). These structures, which are very similar, reveal the presence of five domains in
the protein (Figure 11). The first two domains are homologous to EF-2 while the other
three domains (at the C-terminus of the protein) resemble RNA binding proteins.
Although EF-2 from S. cerevisiae exhibits moderate similarity (~39% homology) to EFG, there appears to be a considerably higher degree of homology between GTP binding
domains of the proteins (Perentesis et. al., 1992).
Recently, a three-dimensional picture of EF-2 in complex with the 80S ribosome
at 17.5 Å resolution has been obtained using cryo-electron microscopy (Gomez-Lorenzo
et. al., 2000). This structure of EF-2 is similar to that of the EF-G70S ribosome
complex (Agrawal et. al., 1998) and the resolution is high enough to allow the tentative
identification of EF-2 domains homologous to those in EF-G. This structure shows that
the domain IV of EF-2 is larger and more complex than its bacterial counterpart, EF-G.
Domain IV of EF-2 mimics the acceptor stem of the tRNA within the ternary
complex EF-2.tRNA.GTP (Avarsson et. al., 1994, Nissen et. al., 1995). Sequence
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25
alignments of EF-2 and EF-G have placed the diphthamide residue at the tip of domain
IV, which roughly corresponds to the position of the anticodon with tRNA. ADPribosylation of EF-2 is thought to interfere with this interaction rendering EF-2 inactive.
1.9
Inhibitors of Poly (ADP-ribosyl) ation
In the past two decades inhibitors of PARP-1 have been used as diagnostic tools
to study the function of ADP-ribosylation reactions that occur widely in biological
systems. In the early 80’s, Purnell and coworkers showed that substituted benzamides
function as potent inhibitors of PARP-1 with a Ki value of approximately 2 M (Purnell
et. al., 1980). Since then, there has been a large body of literature reporting the use of a
variety of substituted-benzamides as inhibitors of PARP-1 function (Tanaka et. al., 1981;
Sims et. al., 1982).
Sims and coworkers, having determined structure-activity relationships of
benzamides and other related compounds, identified the requirements for a compound to
serve as an effective inhibitor of PARP-1 (Sims et. al., 1982). Structure-activity
relationships observed with some of these compounds are presented in Table 1.
Most of the afore-mentioned inhibitors have been found to exert side effects in
vivo rendering an unambiguous interpretation of the observations difficult (Berghammer
et. al., 1999, Ame et. al., 1999). Furthermore the inhibitors of PARP have also been
implicated in affecting other enzymes involved in signaling, namely mono-ADPRTs and
NAD+ glycohydrolases. As a consequence, there is a continuous effort to design more
specific and potent inhibitors of PARP. Among the more recent inhibitors of PARP-1 are
26
the derivatives of 1,8-naphthalimide (Schlicker et. al., 1999), the parent of which is
presented in Figure 12.
The structure of PARP complexed with several inhibitors has also been solved
(Ruf et. al., 1998) and the binding of the inhibitors seems to mimic the nicotinamide
moiety of NAD+. The NAD+ binds PARP in the cleft at the contact of the two central sheets shown in red (Figure 13). The part of the cleft that binds the nicotinamide portion
is very similar to the cleft in both ETA and DT; moreover, the residues involved (Gly
863, Tyr 907, and Glu 988) are conserved. However, the portion of the binding cleft, that
accommodates the adenosine moiety of NAD+, is different in PARP compared to the
toxins. In case of the toxins, this portion of the cleft is shallow and solvent-exposed,
whereas in PARP this site is a pocket lined by helix F of the additional N-terminal
domain.
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28
29
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1.10
Objective
Pulmonary infection in cystic fibrosis (CF) patients is associated with chronic
progressive lung disease (Nicas & Iglewski 1985). CF patients are prone to infection by
P. aeruginosa. Exotoxin A, produced by this organism has been implicated to cause
damage to lung tissue and hence can serve as an excellent target for the development of
new antibacterial agents. Needless to mention that the development of effective
antibacterial strategies depends on detailed knowledge of the structure-function
relationship in ETA.
The current studies were initiated to elucidate the molecular basis for the ADPRT
activity of exotoxin A. Consequently, investigations focused attention towards the
following aspects: (1) development of a fluorometric assay that could provide a rapid,
reliable and sensitive procedure for assessing the kinetic parameters of ETA catalyzed
ADPRT activity; (2) examination of structure-activity relationships of a series of
compounds and identification of the type of inhibition exerted by the parent compound;
and (3) analysis of the interaction of the catalytic domain of exotoxin A with elongation
factor 2 by approaches based on site-directed mutagenesis, chemical modification as well
as fluorescence energy transfer experiments.
31
MATERIALS
2.1
E. coli strains
BB101 (ara (lac pro) nal A arg Eam rif thi-1 F’lac Iq lac+ pro+ sly D) was
generously supplied by Dr. Alan Davidson (University of Toronto, Toronto,
Ontario). MV1190 ((lac-proAB)thi supE44 (Sr1-recA)306::Tn10[F’:traD36
proAB lac Iq (lacZ)M15]) was purchased from Bio-Rad Laboratories (Canada)
Ltd. Mississauga, Ontario. Epicurian coli XL-Blue (recA1 endA1 gyrA96 thi-1
hsdR17 supE44 relA1 lac [F¢proAB lacIqZDM15 Tn10 (Tet)]) was obtained from
Stratagene, La Jolla, CA.
2.2
Plasmid
The vector encoding for PE24 was generous gift from Dr. Ira Pastan and a poly
CAT sequence (6 repeats) was inserted just before the termination codon of the
gene.
2.3
Supplies
ampicillin
Roche Diagnostics
Laval, QC.
dNTPs, Ultrapure
RNase A
T7 Polymerase
DTT
Sigma-Aldrich Canada Inc.
Oakville, ON.
32
CAPS
MOPS
Bis-Tris
DTNB
PMSF
Trizma
NAD
Sigma-Aldrich Canada Inc.
Oakville, ON.
-NAD
-AMP
calcium chloride dihydrate
Fisher Scientific
Toronto, ON
magnesium sulfate heptahydrate
hydrochloric acid
potassium phosphate dibasic
potassium phosphate monobasic
sodium acetate
acetic acid
sodium chloride
sodium sulfate
-mercaptoethanol
EDTA
dextrose
ammonium persulfate
bisacrylamide
Bio-Rad Laboratories (Canada) Inc.
Mississauga, ON.
33
acrylamide
SDS
SDS-PAGE molecular weight standards
Hyroxyapatite
Econo-Pac 10 DG
Agar
BDH Inc.
Toronto, ON
Tryptone
Yeast extract
Dpn I & buffer
Stratagene
La Jolla CA.
pfu polymerase
Rsa I R.E. & buffer
Amersham Pharmacia Biotech Canada
Baie d’Urfe’, QC.
oligonucleotide primers
chelating agarose resin
Q-Sepharose Fast Flow
DNA sequencing
Guelph Molecular Super center
1,5-IAEDANS
Molecular Probes Inc.
Hornby, ON.
5-IAF
Centri-Prep 10
Amicon
Bedford, MA.
Centri-Prep 50
BCA reagents
BSA
Pierce
Rockford, IL.
34
Qiagen plasmid starter kit
Qiagen Inc.
Mississauga, ON.
Sau 96 R.E. & buffer
Promega & Fisher
Nepean, ON.
Guilford Pharmaceuticals
Baltimore, MA
GLFD compounds
35
METHODS
3.1
Molecular biology techniques
The composition of media and buffers used in this section is given in Appendices A and
B.
3.1.1 Preparation of competent cells (Cohen et al., 1972)
The bacterial culture(BB101) was grown at 37° C overnight in 50 mL of SOB
media with constant shaking. One mL of this starter culture was used to inoculate 100
mL of the same media and it was grown to an optical density of ~0.3 (560 nm) at 37° C.
The culture was chilled in an ice bath for 15 minutes prior to centrifugation (2,000 xg) for
20 minutes. The cell pellet was then resuspended in buffer 1 and incubated on ice for 15
minutes. The cell suspension was once again centrifuged (2000 xg) for 15 minutes and
the pellet was resuspended in 8 mL of buffer 2. Aliquots (300 l) were transferred to
sterile microfuge tubes and frozen and stored at –70° C.
3.1.2 Transformation Protocol (Cohen et al 1972)
The desired plasmid (approximately1 g) was added to a suspension of BB101
competent cells (150 l) and incubated on ice for 45 minutes. The mixture was exposed
to 45 seconds (heat shock at 42 C), followed by incubation on ice for 5 minutes. One
mL of YT (2x) was added to the above mixture and incubated at 37° C for 1 hour. A
sample (150 l) of the transformed cell suspension was streaked onto YT (2x) agar plate
36
(Appendix A) containing ampicillin (100 mg/l) and incubated in 37°C for approximately
24 hrs.
3.1.3 Isolation and purification of plasmids
(i) Mini-scale preparation: E. coli BB101 cells, transformed with the plasmid of
interest, were grown in 5.0 mL of YT (2x) medium containing ampicillin (100 mg/l) at
37°C overnight. After ~16-18 hrs the culture was centrifuged (16000 xg) for 5 min. The
pellet was resuspended in lysis (200 l) buffer followed by the addition of alkaline buffer
(400 l) prior to incubation at r.t. for 5 minutes. Next, buffer 3(300 l) was added with
constant mild agitation to the above suspension. The precipitated proteins and
chromosomal DNA were removed by centrifugation (16 000 xg) for 15 minutes and the
clear supernatant was removed from the pellet. Isopropanol (500 l) was added to the
supernatant and incubated at r.t. for 5 minutes to precipitate DNA. The DNA pellet,
following 10 minutes centrifugation (16000 xg) at 4° C, was washed with chilled ethanol
(70%) and dried under vacuum. The dried DNA pellet was then dissolved in 20 l of TE
buffer and stored at –20° C for use when needed.
(ii) Qiagen-mini scale preparation: The plasmid preparations used for nucleotide
sequence analysis were prepared using the Qiagen plasmid kits (refer to Appendix B for
the buffer solutions used) according to the procedures recommended by the supplier.
BB101 cells transformed with plasmid of interest were grown overnight at 37° C in YT
(2x) media (5 mL) supplemented with ampicillin (100 mg/l). The cells were harvested by
centrifugation (16000 xg) for 5 minutes. The pellet was resuspended in buffer P1 (300
l). Following a 5-minute incubation at r.t., buffer P2 (300 l) was added and mixed
gently. This was followed by the addition of buffer P3 to the clear and viscous solution
37
and mixing by inverting the tube. The mixture was incubated on ice for 5 min and the
precipitated proteins and chromosomal DNA were removed by centrifugation (16,000 xg)
for 10 minutes. The clear filtrate was applied onto a Qiagen column equilibrated with
buffer QBT and allowed to enter the column by gravity flow. The Qiagen Tip was
washed with buffer QC followed by elution of the plasmid DNA with buffer QF (1.0
mL). The DNA was precipitated with 0.7 volumes of isopropanol and stored at –20° C
until further use.
3.1.4 Digestion of plasmid DNA with restriction endonucleases
The plasmid DNA was digested with the appropriate restriction endonuclease
(R.E.) using the conditions recommended by the manufacturer. Plasmid DNA (~ 1.0 g)
was used for each reaction. A typical reaction mixture is described below:
H2O
5.0l
buffer, 10 x (appropriate for R.E.)
1.0 l
plasmid ( g/l)
3.0 l
R. E.
1.0 l
The restriction endonuclease used were as follows: (i) the plasmid containing the S449C
mutation was digested with Sau 96 (5 units) for 2 hrs at 37°C; (ii) the plasmids
containing the S459C and S410C mutations were digested with Rsa I (10 units) for 1 hr
at 37° C.
38
3.2
Growth of Escherichia coli and transformants
3.3 Escherichia coli BB101
E. coli BB101 (DE3) [ara(lac pro)nal A arg E am rif thi-1/F’lacIzlac+pro+] was
generously provided by Dr. Alan Davidson from University of Toronto. The cells were
grown and were subsequently maintained as glycerol stocks at –70° C. A typical starter
culture was prepared by inoculating the bacterial cells from frozen stocks into YT (2x)
media (5.0 mL) and grown for 14-18 hrs by incubating at 37° C with continuous shaking.
3.3.2 Growth of organism
E. coli BB101 (DE3) cells were transformed with the plasmid pPE5-399 or its
variants. The starter culture was usually grown in YT (2x) media and then transferred to
super LB (Appendix A). The media was supplemented with 5% dextrose in all cases.
3.4 Site directed mutagenesis of PE24
The individual replacement of serine residues 408, 410, 449, 459, 507, 515, 585,
or glycine 486, and threonine 442 was achieved using either Kunkel’s procedure (Kunkel
1985) or the QuikChange mutagenesis approach developed by Stratagene Cloning
Systems LaJolla, California employing the protocols recommended by the supplier
(Instruction manual, catalog # 200518, Revision #118002).
The Kunkel procedure involves use of a single stranded viral DNA from M13
phage grown in an E. coli dut- ung- strain as the template. Complementary primers
carrying the desired mutation (~25 bases in length), utilize T7 DNA polymerase for the
replication of complementary strand of the template plasmid. This strategy was
39
employed to effect S410C, S449C, and S459C mutations in PE24. In the QuikChange
method, the relatively high fidelity Pfu DNA polymerase is utilized for simultaneous
replication of both strands of the plasmid. This procedure uses double stranded DNA as
the template and two synthetic oligonucleotide primers containing the desired mutation
3.3.1 Designing primers
For the replacement of serine 449 with cysteine, the primer was synthesized with:
(i) a change of the triplet AGC, coding for serine to TGT, the triplet for cysteine; and (ii)
a silent mutation in the triplet coding for alanine 448 from GCG to GCC. The latter
mutation leads to introduction of a unique Sau 96 restriction enzyme site in the parent
plasmid. For the replacement of serine 410 with cysteine, the primer was synthesized
with: a change of the triplet AGC, coding for serine to TGT, the triplet for cysteine
results in the introduction of an additional Rsa I restriction enzyme site in the parent
plasmid. For the replacement of serine 459 with cysteine, the primer was synthesized
with: (i) a change of the triplet AGC, coding for serine to TGC, the triplet for cysteine;
and (ii) a silent mutation in the triplet coding for Val 455 from GTG to GTA. The latter
mutation leads to introduction of a Rsa I restriction enzyme site in the parent plasmid. For
the replacement of theronine 442 with cysteine, the complementary primers were
synthesized with: (i) a change of the triplet ACC, coding for theronine to TGC, the triplet
for cysteine. For the replacement of serine 585 with cysteine, the complementary primers
were synthesized with: (i) a change of the triplet TCC, coding for serine to TGC, the
triplet for cysteine. For the replacement of serine 507 with cysteine, the complementary
primers were synthesized with: (i) a change of the triplet AGC, coding for serine to TGC,
40
the triplet for cysteine. The details of the primers employed in achieving site directed
mutagenesis of the gene encoding for PE24 are presented in Tables 2 and 3.
3.3.2
Mutagenesis by the Kunkel procedure
(a) Phagemid preparation: E. coli strain CJ236 was transformed with plasmid
containing the F+ origin of replication. The suspension was streaked on YT (2x) agar
plates containing ampicillin (100 g/mL) and chroamphenicol (30 g/mL) and incubated
at 37°C for 12-18 hrs. A single colony was selected and allowed to grow at 37°C in
media A (5.0mL) for 12-18 hrs. This starter culture was used to inoculate medium A
(200 mL) supplemented with deoxy uridine (final concentration, 0.5 g/mL) and allowed
to grow to an OD550 of ~0.1 at 37°C. Following addition of helper phage M13K07 (1 mL)
and growth for 1 hr, kanamycin sulfate (50 g/mL) was added and the culture was
allowed to grow for an additional 6 hrs at 37°C. The cells were harvested by
centrifugation (17,000 xg) for 20 minutes at 4°C. The supernatant was carefully
collected and centrifuged as before. RNAse A (10 units) was added to the collected
supernatant and incubated at 25°C for 30 minutes. An aliquot (7.5 mL) of solution
containing PEG (20%) and ammonium acetate (3.5 M) was added to the above mixture
and incubated on ice for 30-60 minutes. The phagemid was collected following
centrifugation (17,000 xg) for 20 minutes and resuspended in salt buffer (Appendix A)
and incubated on ice for 30 minutes. The supernatant following centrifugation (16,000
xg) for 2 minutes was removed and stored at 4°C. DNA from this preparation was
extracted within a few days.
41
42
43
(b) Phosphorylation of primers: The primer containing the desired mutation was
phosphorylated prior to mutagenesis. The following reagents in the amounts indicated
were introduced in the order shown: ATP (1.0 mM), 26 l; 1-phor-all PLUS buffer, 6 l;
water, 60 l; and T4 polynucleotide kinase (3 U/l), 2 l. The reaction mixture was
incubated at 37°C for 45 minutes. Heating the mixture at 65°C for 10 minutes stopped
the reaction. The preparation was stored at -20°C for use as required.
(c) The single stranded DNA, containing the PE24 gene and the ampicillin
resistance gene, served as the template for the T7 DNA polymerase. The annealing of the
primer to the template was accomplished by incubating template (0.3 pmol), primer (6-9
pmol), and annealing buffer (10x) in final volume of 10 l at 85°C and allowing it to cool
to a temperature of 35°C at a rate of approximately 0.1°/min. The following reagents in
the amounts indicated were added to the above mixture in the order shown: T7 reaction
buffer (10x), 2 l; dNTP mix (10x), 2 l; T4 DNA ligase (4 U/l), 1 l; T7 DNA
polymerase (0.2 U/l) 1 l; and sterile water, 4 l. The reaction mixture was subjected to
thermal cycling as follows:
4°C, 5 minutes
25°C, 5 minutes
37°C, 60 minutes
70°C, 10 minutes
Following the temperature cycling, the reaction mixture (15-20 l) was used to transform
MV1190 competent cells (300 l) and the transformants were selected from nutrient agar
plates on the basis of ampicillin resistance.
44
3.3.1 Conditions for Quik Change® mutagenesis
The plasmid pPE5-399, containing the PE24 gene and the ampicillin resistance
gene, served as the template for the Pfu DNA polymerase. The following reagents were
added for the reaction mixture: homogenous pPE5-399 (13 ng/l), 2 l; Pfu reaction
buffer (10x), 5 l; primer 1 (125 ng/l), 1.0 l; primer 2 (125 ng/l), 1.0 l; dNTP mix
(10 mM), 2.5 l; ddH2O, 38.0 l; and Pfu DNA polymerase (2.5 U/l), 1.0 l. This
reaction mixture was kept at 4°C until ready for thermocycling. The temperature cycling
was programmed as follows:
initial period
95°C, 30 seconds
95°C, 30 seconds
55°C, 1 minute
68°C, 8 minutes
The cycle, excluding the initial period, was repeated 12 times followed by a dwelling
time of 15 minutes at 68 C to allow for the extension of any incomplete replications.
Following the temperature cycling, the reaction mixture was treated with 10 units
of Dpn I for one hour at 37° C to degrade the parent plasmid. The mutation-containing
DNA product was used to transform the competent cells of Epicurium coli ®XL-Blue and
the transformants were selected from agar plates based on ampicillin resistance.
3.4
Isolation and Purification of proteins
3.4.1 Overexpression and Purification of WT PE24
The C-terminal fragment of ETA (PE24) was overexpressed in E. coli strain
BB101 (DE3) cells and purified using the protocol of Beattie and Merrill with slight
45
modifications (Beattie et. al., 1995). Two microliters of plasmid pPE5-399, which
contains a repeat of the trinucleotide, CAT, that codes for a poly His sequence at the Cterminus of the PE24 protein, was used to transform BB101 (DE3) cells. The
transformation mixture was plated onto two YT (2x) medium plates containing ampicillin
(100 g/mL) and allowed to grow at 37C overnight. Cells were scraped from plates and
transferred to 50 mL of YT (2x) broth containing ampicillin (100 g/mL). The culture
was grown at 37 C to a high cell density (~ 1h) and aliquots (10 mL) were transferred to
each of two culture flasks containing super L-broth (500 mL) supplemented with MgSO4
(0.4%) and glucose (0.5%). After being grown to an OD650 value between 0.5-0.7, the
cultures were treated with IPTG (1 mM) and grown for an additional 90 min at 37 C.
The cells were collected by centrifugation (10,000 x g) for 10 min and the cell pellet was
suspended in 100 mL of Tris (20 mM, pH 7.9) containing EDTA (10 mM) and sucrose
(20%). The periplasmic fraction was isolated as previously described by Rasper and
Merrill (1994), and the extract was loaded onto a Q-Sepharose Fast-Flow anion exchange
column (10 mL), previously equilibrated in buffer 4 (20 mM Tris.HCl, pH 7.8)
containing NaCl (50 mM). The column was washed with 20-bed volumes of buffer 4 to
ensure removal of EDTA prior to elution with buffer 4 containing NaCl (300 mM). The
effluent was then passed through a 1.0 mL chelate-agarose affinity column (Pharmacia)
charged with 50 mM NiSO4.
The column was washed initially with 5 mL of buffer 5
(20 mM Tris.HCl, 500 mM NaCl, pH 7.9) containing imidazole (5 mM) and
subsequently with 10 mL of buffer 5 at a higher imidazole concentration (60 mM). The
PE24 protein, possessing a poly His tag, was recovered from the column by elution with
8 mL of buffer 5 containing imidazole (250 mM). All fractions (1 mL each) were
46
analyzed by SDS-PAGE and those exhibiting a major band at Mr 28 kDa were pooled,
and dialyzed overnight in buffer A containing EDTA (1mM). The dialyzed sample was
concentrated to 1.0 mL using an Amicon Centriprep concentrator (10 kDa MWCO )
dispensed into small volume aliquots, quick frozen in a dry ice-methanol bath and stored
at –70 C.
3.4.2 Overexpression and Purification of PE24 mutants
The procedure employed for the purification of PE24 single Cys mutant proteins
was identical with that employed in the case of parent protein except for the change
mentioned below. The affinity matrix (Phamacia column, 1 mL in volume) was charged
with ZnSO4 (100 mM) instead of NiSO4 (50 mM). This switch from NiSO4 to ZnSO4
was performed to avoid Ni ion induced modification of the cysteine thiol function of the
protein.
3.4.3 Purification of EF-2 from Wheat germ
Wheat germ served as the source of EF-2 and the procedure employed was
developed in our lab (Armstrong & Merrill 2001). Crude extract from wheat germ
(obtained from New Life Mills, Hanover, ON; 450g) was adjusted to pH 5.0 and the
precipitate formed was collected and dissolved in 2 L of Tris (50 mM, pH 8.1) containing
MgOAc (5mM), CaCl2 (4mM), KCl (100 mM) and -mercaptoethanol (0.7%). The
precipitate formed upon addition of ammonium sulfate (35% saturation) was collected
and re-dissolved in 1.5 L of buffer 6 (25 mM potassium phosphate, KCl 50 mM, pH 7.0)
containing glycerol (5%) and applied to 8 x 15 cm columns of hydroxyapatite
47
equilibrated with buffer 6. The chromatographic mixture was washed with buffer 6
containing potassium phosphate (60 mM) prior to elution with buffer 6 containing
potassium phosphate (300 mM). The protein fraction recovered was subjected to further
chromatography on Fast Flow Q-Sepharose and the protein preparation was concentrated
to (10-20 mg/mL) and stored at -70C.
3.5
Analytical Methods
3.5.1 Determination of protein concentration
The concentration of protein was determined using two methods.
In the first method the absorbance of the PE24 protein (or its variants) was
measured at 280 nm and the protein concentration was estimated using an M value of
27,310 M-1cm-1(Beattie et al., 1996).
In the second method the Pierce BCA protein assay kit was used. In this method
a fresh set of protein standards by diluting the BSA stock (2.0 mg/mL) in the range of 01.2 mg/mL were prepared. The standards and protein samples (10 l) were loaded into a
microwell plate. The BCA working reagent was prepared fresh by mixing 50 parts of
BCA reagent A with 1 part of BCA reagent B and an aliquot (200 l) was added to each
well. The plate was then covered and incubated at 37°C for 30 minutes. After
incubation, the plate was cooled to room temperature and absorbance was measured at
562 nm on plate reader. A standard curve was constructed by plotting the A562 reading
for each BSA standard (corrected for blank) versus its concentration in mg/mL. Using
the standard curve, the protein concentration of PE24 or its variant(s) as well as EF-2 was
determined.
48
3.5.2 Determination of homogeneity and molecular weight of the protein preparations
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE
(Laemmli 1970), was used to assess the homogeneity as well as to obtain an estimate of
the molecular weight of the purified PE24 preparations. The content of acrylamide in the
stacking and separating gel was 3% and 12.5%, respectively. The gel was stained with
Coomassie Blue R-250 after electrophoresis under constant current of 13.0 mA. A
solution of isopropanol: acetic acid: water (30:10:60) was used for destaining purposes.
For a more accurate determination of molecular weight of the protein preparations
electrospray mass spectrometry was used (Siuzdak, 1994). Protein samples were
dialyzed in ammonium bicarbonate buffer prior to analysis by mass spectrometry.
3.5.3 Reaction of PE24 with 5-[[[2-iodoacetyl]amino]ethyl]amino]-naphthalene-1
sulfonic acid (IAEDANS)
Each of the PE24 variants required different conditions in order to achieve a high
degree of modification. The following procedure describes the general steps in the
modification reaction and the change in the conditions for each variant is listed in Table
4. Preparations of PE24 cysteine muteins (0.5 mg) in 200 mM Tris.HCl, pH 8.1 were
treated with excess DTT at r.t. for 15-60 minutes. Following this incubation, an aliquot
of IAEDANS (5 mM) was added to the reaction mixture to achieve 10-50 fold excess of
the reagent over the thiol content of the protein and DDT. The reaction mixture was
allowed to mix on a nutator at r.t. for 1-4 hrs. The excess IAEDANS was quenched by
the addition of DTT (100 mM) to the reaction mixture.
49
50
The labeled protein was recovered free of the extraneous materials by
chromatography on a BioRad gel filtration column (10G) with 20 mM Tris, pH 7.9
containing NaCl (50 mM) serving both as equilibration as well as elution medium. An
 value of 6,000 M-1cm-1 at 337 nm (Molecular Probes catalog) for the chromophore
incorporated into PE24 variant(s) was employed for assessment of the labeling efficiency.
Calculations based on absorbance at 337 nm for modified PE24 variants indicated
labeling efficiency in the range of 80-100%. The only exception being the PE24 variant
with the label located on the amino acid residue 408 which was labeled to ~60%.
3.5.4 Preparation of fluorescein-labeled EF-2
EF-2 (104 M) in 500 mM Tris.HCl, pH 8.0, was treated with an equimolar
amount of 5-iodoacetamido-fluorescein. After 5 min at 25 ºC the reaction was terminated
by the addition of DTT (final concentration 50 mM). The labeled EF-2 was recovered by
chromatography on a BioRad Gel filtration column with 20 mM Tris, 150 mM KCl, pH
7.9 serving as equilibration and elution medium. Fractions containing the protein were
concentrated using Centri-Prep concentrator (Amicon, MI). An M value of 70,000 M1
cm-1 (492 nm) for 5-AF (Molecular Probes catalog) was employed for estimating the
labeling efficiency. Estimates based on the absorbance at 492 nm and the corresponding
protein concentration indicated a labeling efficiency of approximately 12%. Since there
are 8 cysteine residues in EF-2, only one of the 8 was accessible to alkylation by the
fluorophore under these reaction conditions.
51.
3.5.5 Reaction of PE24 with DTNB
The thiol content in the PE24 Cys mutant proteins was assessed by reaction with
DTNB (Ellman 1959). In a typical experiment, protein (30-60 M), in one mL of
potassium phosphate (200 mM, pH 8.0), was treated with an aliquot (250 l) of aqueous
solution of DTNB (2 mM) and the increase in absorbance at 412 nm was recorded. The
above reaction, performed in the absence of the protein, served as control and allowed for
subtraction of the contribution due to spontaneous hydrolysis of the reagent. An M
value of 14,150 M-1cm-1 (Riddles et al., 1979) was used to determine the number of thiol
groups present in the protein.
3.5.6 Determination of ADPRT activity
(i) ADPRT assay: A typical assay, in a final volume of 70 l, consisted of NAD+ (desired concentration), EF-2 (20 M) and buffer (20 mM Tris.HCl, pH 7.8) in a 3
mm pathlength ultramicrocuvette (Helma Inc., Concord, ON) placed in the sample
chamber of the fluorometer. Following temperature equilibration for 10 min, the reaction
was initiated by the introduction of PE24 (5 nM). The reaction was monitored by
recording the increase in fluorescence intensity with time (excitation 305 nm, emission
309 nm cut-off filter). These experiments were performed over a wide range (0-700 M)
of -NAD+ using appropriate aliquots of a stock solution of this compound (57 mM) in
above buffer. In experiments where EF-2 concentration was varied, the protein was used
over the range of 2-25 M. In all experiments the KCl concentration was kept at 50 mM
concentration.
52.
(ii)-AMP Standard Curve and Assay Calibration: A stock solution of 44.7 mM
-AMP in distilled water (M265 = 10,000 M-1cm-1) was used to prepare a series of
samples in 20 mM Tris, pH 7.8 over the concentration range of 0 - 7 M. The
fluorescence emission of each of the samples was measured (excitation 305, emission
309 nm cut-off filter). A standard curve constructed from the data was used in the
assessment of the rate of enzyme catalyzed reaction.
3.5.7 Determination of NAD binding
The NAD-dependent quenching of intrinsic protein fluorescence was monitored
as a function of its concentration as previously described (Beattie et. al., 1996). Samples
of toxin (1.25 M) in 600 l of 20 mM Tris.HCl, pH 7.9 containing NaCl (50 mM) were
treated with NAD+ over a range of 0-800 M. Samples were excited at 295 nm (4 nm slit
width), and the fluorescence intensity was recorded at 340 nm (4 nm slit width) for 30
seconds in a computer-controlled PTI alphascan-2 spectrofluorometer (Photon
Technology International Inc.).
3.5.8 Differential scanning calorimetry (DSC)
DSC measurements for PE24 were carried out with the differential scanning
microcalorimeter (Microcal-2) over the temperature range of 5-45 C (heating rate, 1 
per min; and excess pressure, 20 psi). The protein concentrations ranged from 0.5-2.0
mg/mL in a sample of 1.5 mL in volume. Protein solutions for DSC measurements were
prepared by dialysis against a 20 mM Tris, pH 7.9 buffer for PE24 and 20 mM Tris, pH
7.9 containing 300 mM KCl and glycerol (5%) for EF-2 samples at 4 C. Primary data
53.
processing was carried out using the Origin software (Microcal, Inc., Northampton, MA).
The Nano II Differential Scanning Calorimeter (N-DSC II) (Calorimeter Sciences
Corporation, Spanish Fork, UT) at the University of Waterloo was used to obtain the
melting temperature of EF-2.
3.6 Fluorescence Methodologies
3.6.1 Steady-state fluorescence
Emission and excitation spectra were obtained using a computer-controlled PTI
Alphascan-2 spectrophotometer (Photon Technology Inc.). All measurements were
conducted at 25°C unless otherwise indicated. All spectra were recorded with 4 nm slit
width for both excitation and emission. For all fluorescence measurements, a wedge
depolarizer was placed on the exit side of the excitation monochromator, and emission
was detected at right angles. Wavelength dependent bias of the optical and detection
systems was corrected, and appropriate blanks were subtracted.
3.6.2 Anisotropy measurements
Polarization measurements on 5-AF and 1,5-AEDANS labeled samples were done
using 492 nm, and 337 nm excitation, respectively. The measurements were performed
using Glan Thompson prism polarizers and a “T-format” detection system with the
excitation being vertically polarized. The 5-AF labeled EF-2 was excited at 492 nm (slit
width 4 nm) and emission was measured at 500 nm (slit width 4 nm). The 1,5-AEDANS
labeled PE24 variants were excited at 337 nm (slit width 4 nm) and emission was
measured at 450 nm. The “G” instrumental factor, IHV/IHH, was determined using
horizontally polarized excitation. The anisotropy was calculated from the respective
54.
horizontal and vertical fluorescence emission components of a measured 30 seconds scan,
which was averaged. Each anisotropy measurement is the average of 2 separate
experiments done in triplicate. The equation for calculating anisotropy is
R
bg bg
bg bg
I VV  G. I VH
I VV  2G. I VH
Where VV corresponds to vertical polarization of excitation beam and vertical
polarization of emission of the sample, VH refers to vertical polarization of excitation
beam and horizontal polarization of emission. The G factor is the instrument correction
factor accounting for the differences between the monochromators.
3.6.3 Fluorescence Resonance Energy Transfer Assay
The efficiency of FRET between the donor and acceptor can be written as:
E = 1- (F/Fo)
Where F and Fo are the fluorescence intensities of the donor in the presence and absence
of the acceptor, respectively. Fluorescence resonance energy transfer using the PTI
instrument as outlined above was conducted to monitor EF-2/PE24 complex formation.
The parameters were set for detection of 1,5-AEDANS probe where the sample (1.0 M)
was excited at 337 nm (slit width 4 nm) and emission was measured for 30 seconds at
460 nm (slit width 4 nm) using microcuvettes. The PE24 sample in Tris buffer (20 mM,
50 mM NaCl, pH 7.9) was titrated with increasing amount of EF-2AF (0-5 M). All
measurements were corrected for background fluorescence intensity.
55.
3.7
Miscellaneous Procedures
3.7.1 Effect of KCl on ADPRT activity of PE24
The ADPRT activity of PE24 was measured at varying KCl concentrations
ranging from 50-800 mM (Vmax conditions). The concentrations of both -NAD+ and EF2 substrates were kept constant at 500 M and 20 M, respectively. All measurements
were performed at 25 C.
3.7.2 Effect of temperature on ADPRT activity of PE24
For assessing the effect of temperature on the enzyme activity of PE24, the
ADPRT reaction was performed over a temperature range of 0-40 C in 5 C increments
with the aid of a digital thermistor with a fine wire thermocouple that bathed in a
reference cuvette filled with water in the multi-cell holder of the fluorometer. The
reaction mixture was incubated at each temperature for 10 minutes prior to the initiation
of the reaction by the addition of the toxin.
3.7.3 Effect of pH on ADPRT activity of PE24
The pH versus rate profile was obtained by measurement of catalytic activity over
the pH range of 2-11. Buffers employed were: sodium acetate, pH 2-4; bis-TRIS, pH
4.5-7.0; Tris-HCl, 7.4-8.5; and CAPS, pH 9-11. All the measurements were recorded at
25 C. Spontaneous hydrolysis of -NAD+ at each pH was deemed not significant
enough to interfere with the ADPRT reaction.
56.
3.7.4 Effect of KCl on NAD binding of PE24
The effect of KCl concentration on the binding of NAD+ was examined by
varying KCl in the buffer over a range of 50-600 mM. In a typical experiment, PE24
(1.25 M) in Tris buffer (20 mM, pH 7.9) containing the desired KCl concentration was
titrated with increasing amount of NAD+ (0-900 M). The fluorescence intensity at 340
nm emission (4 nm slit width) was measured for 30 seconds after addition of each aliquot
(2 l) of NAD+.
3.7.5 Effect of KCl on EF-2 binding of PE24
The effect of KCl concentration on binding of EF-2 to PE24 was measured by
varying its concentration (0-600 mM) in the binding buffer. The procedure was identical
to the one outlined in section 3.7.3 except that the amount of KCl in the buffer was
adjusted to obtain the desired concentration.
3.7.6 Effect of various ions on EF-2 binding of PE24
The ADPRT activity of PE24 is very sensitive to the concentration of KCl in the
reaction mixture (see Results section page 73). Hence, an investigation of the effects of
various anions (SO42-, F-, or Cl-) on the binding of EF-2 to PE24 was undertaken. For
these experiments the fluorescence energy transfer of IAEDANS to fluorescein was
measured (as described in section 3.7.3) using protein solutions in the desired buffer.
Thus, in the experiments used for examining the effect SO42- ions, Tris buffer (20 mM,
pH 7.9) was supplemented with Na2SO4 over the range of 50-400 mM. In case of F- ions,
57.
the reaction mixture consisted of Tris buffer (20 mM, pH 7.9) with concentration of NaF
ranging from 50-400 mM.
3.7.7 Effect of temperature
The effect of temperature on binding of EF-2 to PE24 was measured over the
temperature range of 5°C-45°C. The procedure was similar to that outlined in section
3.7.3. After each addition of AF-EF-2, the mixture was incubated at the desired
temperature for 10 min prior to measurement of fluorescence intensity.
3.7.8 Dose-response determination of naphthalimide compounds
The ADPRT activity of PE24 in presence of each of the inhibitors was measured
by the procedure as that discussed in section 3.5.5. In these studies the -NAD+, EF-2
concentrations in the assay medium were held at 500 M, 20 M respectively. The
reaction was initiated by the addition of PE24 (5 nM), which had been pre-incubated (10
min) with the desired concentration of inhibitor (0-100 M). The data were analyzed by
nonlinear regression curve fitting using Origin 6.1 (Micro Cal) with the aid of the
equation
Y = A1 (A2-A1/1 + 10 ([I]-log[IC50])
where Y is the observed activity measured in the presence of inhibitor, and [I] is the
concentration of inhibitor. A1 is loss of activity in the presence of very high
concentration of inhibitor, and A2 is the activity in the absence of inhibitor. IC50 is the
concentration of inhibitor that reduces the activity of the enzyme by 50%. DMSO, which
was present at a final concentration of 0.2% in the assays with the inhibitors had no
58.
adverse effect on ADPRT activity of PE24. All experiments were carried out at least two
times, in triplicate.
3.7.9 Determination of dissociation constant(s) of PE24.Naph and analogues
The quenching of the fluorescence of the tryptophan residues in PE24 was used to
determine the binding constant (KD) for each of the inhibitors. The quenching of PE24
fluorescence was measured in the presence of inhibitor(s). In a typical experiment, the
reaction mixture, in a volume of 70 l consisted: Tris.HCl (20 mM, pH 7.9) containing
NaCl (50 mM), PE24 (0.6 M) and the inhibitor (concentration as indicated). The
quenching of PE24 fluorescence was recorded (excitation, 295 nm and emission 305-450
nm) using a computer-controlled PTI Alphascan-2 spectrofluorometer (Photon
Technology International Inc.) The inhibitor concentration, in all cases except in the case
of GLFD 09 was varied over the range of 0-5 M. Since GLFD 09 was found to function
as a weak inhibitor, a higher concentration range (0-500 M) was employed.
3.7.10 Dialysis experiments
An aliquot of Naph (9.0 mM) in DMSO-ethanol mixture was added to a sample of
PE24 (17.5 M) in 20 mM Tris, pH 7.9 containing 50 mM NaCl to achieve a 100 fold
molar excess of the inhibitor over that of the protein. This concentration of Naph resulted
in approximately 80% loss in PE24 ADPRT activity. A sample of PE24 treated under
identical conditions but with the omission of Naph served as control. The ADPRT
activity of each of these samples both prior to and as well as subsequent to dialysis for 4
days at 4 C was recorded. The concentration of the protein in the samples was
59.
determined subsequent to dialysis so as to make appropriate correction for dilution, a
phenomenon accompanying the dialysis procedure.
3.7.11 NMR experiments
O
H
NH2
O
O
N
O
N
H
CH3
+
N
H
NATA
1,8-naphthalimide
A sample of N-acetyltryptophanamide (NATA) (9 M) in DMSO-d6 was added to
a solution of Naph (9 M) in DMSO-d6 to achieve a 1:1 ratio of the two reactants. The
reaction mixture was mixed at r.t. for 30 min prior to analysis by 1H NMR (400 MHz,
DMSO), : 1.76 (s, 3 H); 2.80-2.92 (dd,1 H); 3.02-3.07 (dd,1 H); 3.33 (s, 2 H), 4.30 (m,
1H); 6.95-7.11 (m, 3H); 7.29-7.31 (d, 1 H); 7.42 (s, 1 H); 7.58-7.60 (d, 1 H); 7.83-7.87 (t,
2 H); 7.95-7.98 (d, 1 H); 8.43-8.48 (t,3 H); 10.78 (s, 1 H).
3.7.12 ESMS analysis
An aliquot of Naph (4.4 mM) in DMSO-ethanol mixture was added to a sample
(300 l) of PE24 (40.8 M) in 20 mM Tris, pH 7.9 containing 50 mM NaCl to achieve
60.
100 fold molar excess. The mixture was lyophilized prior to analysis by electrospray
mass spectrometry.
3.7.13 Kinetic Analysis of the reaction of PE24 with Naph
ADPRT activity of PE24 was determined in the presence of different
concentrations of Naph. The assay in a final volume of 70 l consisted: 20 mM Tris, pH
7.9 containing NaCl (50 mM), EF-2 (20 M), -NAD+ (0-500 M). The assay was
initiated by introducing PE24 (40 nM).
The concentration of Naph when present in the assay was varied over the range
(0-300 nM). A stock solution (90 mM) of the desired inhibitor was prepared in DMSO.
Prior to use in the assay, this stock solution was diluted with ethanol to achieve a final
concentration of approximately 900 M. This solution was further diluted with buffer
(20 mM Tris.HCl pH 7.9 containing 50 mM NaCl) to achieve the concentration used in
the assay. Such an elaborate dilution protocol was employed to avoid precipitation of the
inhibitor.
3.7.14 Molecular Modeling
The molecular modeling package Sybyl®(version 6.7, Tripos Inc.) was used on a
Silicon Graphics O2 R10000 workstation for all molecular modeling studies presented in
this thesis. Web Lab Pro 3.7 was used for visualization and analysis of structures and for
preparing all the modeling figures.
The full coordinates of the catalytic domain of ETA /-TAD complex (Li et al.,
1996) (PDB entry, 1AER), the whole ETA (Allured et al., 1986) kindly provided by Dr.
61.
McKay, DT (Bell & Eisenberg, 1996) (PDB entry, 1TOX), PARP/inhibitor complex (Ruf
et al., 1998) (PDB entry, 2PAX) were obtained from the Brookhaven Protein Data Bank
(PDB). The structures of compounds, Naph and AEDANS, were built using the Sketch
function within Sybyl and were then geometry optimized using the Tripos force field.
The structure of 1,8 naphthalimide was then superimposed onto -TAD using the Fit
Atom command and selecting the atoms of the nicotinamide ring as the point of reference
for the least squares fit. Each of S449C, T442C, and G486C variants of the catalytic
domain structure were generated using the Mutate command and the AEDANS structure
was attached to the Cys residues using the Create Bond command.
Following the docking procedure, the geometries of the models were optimized
using the anneal function. The standard Sybyl energy minimizer, MAXIMIN2, was used
under the following (default) conditions. If the force on any atom exceeded 1000
kcal/mol Å2 on atom-by-atom Simplex minimization was performed until all forces fell
below this threshold. After this threshold conjugate gradient minimization proceeded
until the convergence criteria on an rms force over all atoms of less than 0.05 kcal/mol Å1
was reached. Non-bonded interactions between substructures (residue) more than 8.0 Å
apart were neglected. The electrostatic interactions were included based on partial
atomic charges assigned empirically by the “Gasteiger-Huckel” method (Gasteiger et al.,
1980) in all of the minimizations.
The use of the anneal function allows the MAXMIN2 energy minimization of a
subset region around the site where the inhibitor is bound to the protein. There are three
distinct regions of the protein defined during an anneal minimization. The “hot” region is
defined as a region including all residues 6 Å around the bound inhibitor. The inhibitor
62.
and the amino acid residues in the “hot” region were energy minimized through
conformational and geometrical changes. The “interesting” region contains all of the
amino acid residues within 12 Å of the “hot” region. This region is not subject to energy
minimization during the anneal process, but the effect of the atoms of the “interesting”
region on the atoms in “hot” region is considered during annealing. The third region was
defined as all of the amino acid residues outside of the “hot” and “interesting” regions.
This region is held static during the minimization and is ignored during computations.
63.
RESULTS
4.1 Fluorescence-Based Assay of ADPRT Reaction
In the ADPRT catalyzed reactions, NAD+ normally serves as the source of ADPR
moiety that is used to effect modification of the targeted acceptors. Although the
radioactively labeled NAD+ has been employed routinely for assessing the kinetic
parameters of the reactions catalyzed by ADPRTs, the procedure is time consuming and
laborious. Hence, the possibility of developing a rapid and reliable assay for ADPRT
activity of PE24 using -NAD+, a fluorescent analogue of the diphospho-pyridine
nucleotide was examined in view of the relatively higher quantum yield of the product
formed during the reaction (Barrio et al., 1972). Thus, the ADPRT-catalyzed reaction
can be followed by monitoring the increase in the fluorescence during the course of the
reaction and through the use of -AMP as a standard to convert ADPRT measurements to
catalytic rates with units of M -NAD+ per second (Figure 14).
4.1.1 Physico-chemical properties of PE24 and EF-2
SDS PAGE analysis revealed that the PE24 preparation purified by affinity
chromatography was homogenous (Figure 15). ESMS analysis indicated a molecular
weight of 24530 ±5 Da (Figure 16), a value in accordance with the deduced value based
on its amino acid sequence.
SDS-PAGE analysis of wheat germ EF-2 revealed the preparation to be relatively
free of other extraneous proteins. The molecular weight of EF-2 was estimated to be
approximately 96,000 kDa.
64.
65.
66.
67.
68.
Denaturation thermograms of PE24WT and EF-2 are presented in Figure 17.
Thermal denaturation of both proteins is not reversible under the conditions employed as
the reheating cycle of the protein back through its transition temperature indicated
(Figure 17, dotted line). The Tm of PE24 is at 304K, which indicates the protein is
thermolabile being susceptible to denaturation under physiological conditions. In
contrast, EF-2 is relatively more stable as indicated by a Tm value of 331K.
4.1.2 ADPRT reaction catalyzed by PE24
The dependence of the PE24-catalyzed ADPRT activity on the concentration of
both substrates -NAD+ and EF-2 was investigated. A plot of velocity versus substrate
concentration for both substrates was found to fit Michaelis-Menten kinetic model
(Figure 18). These data provide the basis for the assessment of the kinetic parameters,
KM, Vmax, and kcat for both substrates presented in Table 5. The kcat values were found to
be 675  85 min-1 and 734  67 min-1 for NAD+ and EF-2, respectively. The kcat/KM
values (2.5 x 106 and 92.8 x 106 M-1min-1, respectively) indicate that PE24 fragment is a
moderately efficient enzyme. Substrate inhibition of the ADPRT activity for PE24 was
observed at concentrations of NAD+ >800 M (data not shown).
4.1.3 Effect of pH on the stability and ADPRT activity of PE24
Previous studies had indicated that PE24 was similar to its parent protein in its
catalytic function (Beattie et al., 1996). However, a comprehensive study of the physicochemical properties of PE24, especially the factors influencing its catalytic function still
remained to be achieved.
69.
70.
71.
72.
The pH dependence of ADP-ribosylation activity of PE24 showed a typical bell
shaped curve, with maximal activity at pH ~7.8 (Figure 19A). The effect of pH upon the
measured kinetics parameters, kcat and kcat/KM of PE24 is shown in Figure 19B. The
toxin is most stable at neutral pH and remains in solution when incubated in buffer at pH
values between 5 and 11. Although both substrates, NAD+ and EF-2, are stable over the
wide pH range studied (Beattie et al., 1996) EF-2 was observed to precipitate out of
solution at pH values close to its isoelectric point of 5.3. The Vmax was maximal at pH
7.8, which is in close agreement with the previously shown data using radiolabelled
NAD+ substrate (Beattie et al., 1996). Notably, the KM for the NAD+ substrate was very
sensitive to the pH of the solution. At acidic pH values, the KM was considerably smaller
than in physiological pH buffer whereas at alkaline pH values, the KM was the highest,
showing a general trend of increasing with pH. A plot of kcat/KM as a function of pH
(Figure 19B) exhibited a relatively sharp profile centered at pH 7.8.
4.1.4 Effect of temperature
The ADPRT activity of PE24 was determined at several temperatures over the
range of 5 º to 40º C (see methods, page 54 for experimental detail). As shown in Figure
19, the rate of PE24 catalyzed ADPRT reaction increases steadily as the temperature is
raised from 5º to 30º C and this is followed by a precipitous drop in activity occurring at
30.8º C. This apparent loss of activity at temperatures > 30.8º C would appear to be a
consequence of the propensity of PE24 to undergo thermal denaturation at temperatures
>31º C as indicated by the DSC profile (Tm= 304K) of the protein (Figure 16). An
Arrhenius plot (not shown) of the above data yielded an Ea value of 39  0.5 KJ/mol.
73.
74.
4.1.5 Effect of ionic Strength
Previous studies have shown that the EF-2 protein (substrate) is not stable in
media of low ionic strength (Prentice & Merrill 1999). In addition, the activity of
exotoxin A appears to be influenced by salts present in the buffer (Carroll & Collier
1988). Consequently, the ADPRT activity of PE24 was determined at various KCl
concentrations in the assay. These studies revealed a progressive decrease in PE24
ADPRT activity with increasing concentration of KCl and the activity was virtually
abolished at KCl concentrations of >800 mM (Figure 20). A fifty percent drop in
ADPRT activity was observed at KCl concentration of 150-180 mM.
4.1.6 Effect of ionic strength binding of NAD+
The ability of NAD+ to quench the intrinsic fluorescence of the parent toxin and
its catalytically functional C-terminal fragment PE40 has been determined previously
(Beattie et al., 1999; 1996). NAD+ was found likewise to quench the intrinsic
fluorescence of PE24 (20% reduction) at high concentration of the pyridine nucleotide
substrate. KCl appeared to inhibit this quenching of fluorescence by NAD+ indicating the
ability of salt to promote its binding to PE24 (Figure 22). Thus KCl appears to have an
adverse effect on the KD of the EF-2.NAD+ complex.
75.
76.
77.
4.2
Site-directed Mutagenesis of PE24
The ETA catalyzed ADPRT reaction involves the participation of NAD+ and EF-
2 as substrates. Thus, ETA has to interact with both NAD+ and EF-2. Concerning the
former aspect, considerable information has been recorded in the literature (Domenighini
et al., 1991; 1994; Wick et al., 1990). In contrast, there is a lack of consistency of data
regarding the interaction between EF-2 and the toxin (Collier et al.1988; Elzaim et al.,
1998)
Although fluorescence spectroscopy is an effective tool for studying the structure,
local environment and dynamics of proteins, it could not be used directly in the case of
the toxin-catalyzed reaction in view of one of its substrates, EF-2, also being a
polypeptide. Hence, site-directed mutagenesis of PE24 was undertaken with the aim of
introducing amino acid residue(s) that could serve as carrier(s) for reporter probes.
Cysteine was the amino acid of choice for the following reasons: (1) this amino acid is
not present in the active site domain of the protein; and (2) its thiol function can serve as
a carrier of the reporter probe.
Based on the available data from the three dimensional structure of ETA, two sets
of amino acid residue(s) were selected for replacement by cysteine. The first groups
were those in the vicinity of the NAD+ binding region so as to gain insight into the
interaction between the toxin and its substrates (NAD+ and EF-2). The other set of amino
acids chosen for replacement with cysteine were far removed from the NAD+ binding
region so as to monitor the interaction between the toxin and EF-2. Figure 22 shows the
3D structure of PE24 and the position of each of the residues selected for replacement in
this study.
78.
79.
80.
4.2.1 Characterization of PE24 variants
The replacement of the desired amino acid residue in PE24 with cysteine was
achieved by site-directed mutagenesis as described in the Methods (Page 37). The
success in such amino acid replacement was confirmed by the determination of the
nucleotide sequence of the gene encoding for the protein. Furthermore, the molecular
weight of each of the PE24 variants determined by ESMS was consistent with that
expected on the basis of the amino acid replacement.
Despite this supporting evidence for the incorporation of a cysteine residue in
each of the PE24 variants, DTNB titration failed to reveal the presence of a thiol function
in these preparations. The possibility of oxidation of cysteine residue in PE24 variants
resulting in the formation of dimeric species (intermolecular disulfide bridge) was
evident since both SDS-PAGE and ESMS analysis indicated that under native conditions
a significant amount of dimeric form of the proteins was present.
Since PE24 and its muteins were purified using nickel-agarose affinity matrix, the
possibility that this divalent cation promoted the oxidation of cysteine residue in the
muteins was also explored. Replacement of Ni2+ with Zn2+ in the affinity matrix did not
result in any significant change in protein recovery, but still showed the absence of a
DTNB titratable thiol function.
The above observation suggested that the single Cys residue within PE24 variants
was incapable of reacting with DTNB. A possible explanation for this phenomenon may
be related to PE24 being transformed to a “metallo-protein” upon acquisition of a
cysteine residue. Thus, the newly introduced cysteine residue, in concert with the other
residues such as histidines(s) and/or dicarboxylic amino acid (aspartate or glutamate)
81.
could serve as a tight divalent cation-binding site in the protein. The affinity for the
divalent cation (Ni2+ or Zn2+) would be such that it can not be readily removed by agents
like EDTA, a situation that is analogous to that noted in dinuclear metallo -lactamases
(Scrofani et al., 1998).
The failure to detect any divalent metal ion in ESMS analysis of these PE24
variants might be due to its loss under the experimental protocol which, is accompanied
by denaturation of the proteins(s). Thus, ESMS analysis would provide molecular weight
of protein free of the metal ion.
A cysteine residue was introduced into PE24 so that it can serve as the functional
group for alkylation with IAEDANS and thus serve as a carrier for the reporter probe.
The inability of the parent PE24 to be modified by IAEDANS prompted this approach
since this catalytic fragment is devoid of Cys. Hence, it was surprising to find that PE24
muteins containing single Cys replacements were devoid of DTNB accessible thiol
function, yet they were readily amenable for modification by IAEDANS. However,
DTNB has been shown by other groups to result in ambiguous findings in regards to the
number of accessible thiol groups in proteins. For example, Saccharomyces cerevisiae
dehydrogenase, which possess 4 Cys residues, reacts with DTNB only to the extent of
2.5-3 thiol equivalents under denaturing conditions (Dmitrienko, 2001). On the other
hand, it is possible that the reason for the lack of reactivity of PE24 variants containing
Cys residue towards DTNB, and yet the susceptibility to modification by IAEDANS, is
that the modification by IAEDANS took place at a site other than the desired Cys residue.
Although the probabilities of such an outcome are low, it is still one of the shortcomings
associated with chemical modification of proteins approach and one that we have
82.
considered. If indeed the alkylation has taken place on a site other than the desired
sulfhydryl site, one can rationalize that the introduction of a cysteine residue (by sitedirected mutagenesis) in PE24 is accompanied by conformational change(s) such that
functional group(s) of PE24 becomes amenable to alkylation with IAEDANS. Thus, the
susceptibility of PE24 muteins to modification by IAEDANS would appear to be a
fortuitous event, which helped overcome the problem, created by the loss of reactivity of
the thiol function to DTNB. It is important to note that the main objective of this
approach was to develop a method in which the interactions between the two proteins
could be measured directly and topographical mapping of the interactions that would
require additional site-specific information is in the scope of future work.
4.2.3 Characterization of AF-EF-2
EF-2 preparations were subjected to modification with IAF as described in
Methods (Page 49). Since there are 8 cysteine residues in EF-2, one would expect
alkylation of the thiol functions of some of these residues. The UV-VIS absorption
spectrum of AF-EF-2 is characterized by two absorption maxima at 280 nm and 515 nm.
The absorbance value at 515 nm of the protein sample and its concentration (determined
by BCA procedure, methods page 46) provided the basis for assessing the degree of
alkylation.
The structural integrity of the muteins was analyzed by NAD+ binding and
ADPRT activity of each toxin variant. All PE24 muteins, like the parent protein, possess
ADPRT activity. The cysteine replacements in PE24 did not lead to any significant
change in the affinity for NAD+. Thus, in the case of all nine mutants, the Kd values
83.
range between 35-129 M, which is similar to that recorded in the case of the parent
PE24. The ADPRT activity and NAD+ binding affinity of parent PE24 and its muteins
are presented in Table 6. The ADPRT activity of most of the mutants did not differ
significantly from that of the parent PE24.
4.2.4 Characterization of PE24-AEDANS adducts
Since all the mutants had nearly WT ADPRT activity they were chosen for further
studies. Treatment of the PE24 Cys variants with 1,5-IAEDANS resulted in alkylation of
the thiol functionality in. The fluorescence of IAEDANS is quite sensitive to its
environment and its adducts respond to ligand binding by undergoing spectral shifts and
changes in fluorescence intensity. Because of the large Stokes shift (Hudson & Weber
1973) associated with IAEDANS and an emission that overlaps well with absorption of
other fluorescent dyes such as fluorescein, it is ideal for FRET experiments of
approximately up to 60 Å (Lackowicz, 2000). The alkylation of PE24 variant(s) is
indicated by the appearance of absorbance around 337 nm. Figure 24 illustrates the
absorbance and fluorescence spectra of the parent PE24 and its AEDANS derivative. All
the AEDANS-derivatized PE24 mutants were tested for ADPRT activity and NAD+
binding affinity. As shown in Table 7, there was very little alteration in the ADPRT
activity of most of the cysteine mutants following alkylation. The AEDANS-T442CPE24, AEDANS-S449C-PE24, and AEDANS-G486C-PE24 were among the variants
that showed significantly lower ADPRT activity upon alkylation. The loss of activity
was measured to be 84, 16, and 12 fold less, respectively.
84.
85.
86.
87.
The binding affinity of most AEDANS derivatives of PE24 for NAD+ was
comparable to that of the WT. The AEDANS-S449C-PE24, however, showed
approximately a 10-fold reduction in NAD+ binding affinity, which could account for the
observed reduction in ADPRT activity of this adduct. The AEDANS-T442C-PE24, on
the other hand had about 100-fold lower affinity for NAD+ than WT. The binding
affinity of AEDANS-E486C-PE24 for NAD+ was not significantly altered, which implies
that loss of activity of the alkylated derivative might be due to other interactions.
Since the residues 442, 449 and 486 surround the active site of the enzyme,
molecular modeling studies were carried out to further assess the impact of the dansyl
moiety of the reporter fluorophore on the accessibility of the binding site of the enzyme
to its substrate NAD+. The structure of AEDANS was determined using Sybyl 6.7® and
was energy minimized using the Tripos forcefield and bonded to the sulfur atom of each
of the prospective cysteine residues in the x-ray structure of ETA--TAD complex (Li et
al., 1996). The geometries of these hypothetical complexes was optimized using the
anneal function within Sybyl. This routine is a molecular mechanics method performed
on a defined subset of the substrate-protein complex and generates a local energy
minimum geometry in the vicinity of the introduced probe structure.
In the models, which emerged from the above analysis, the fluorophore was
observed to partially block the access to the NAD binding pocket in the case of the
T442C variant. In the case of both S449C and E486C, the AEDANS moiety was situated
quite close to the NAD+ binding site; however, no true obstruction of the binding site was
observed.
88.
4.2.5 FRET-based binding assay
Currently there is limited information available on the interactions between PE24
and its protein substrate EF-2. Due to the lack of a high resolution structure for EF-2,
there is no direct 3D model of the complex between PE24 and EF-2. Mutational
experiments have indirectly implicated residues such as His 426 to be critical in binding
between the two proteins. Kessler and Galloway proposed that His 426 within ETA is
essential for the interaction of toxin with EF-2. Several researchers have also shown that
mutation of His 426 to Tyr results in loss of binding of PE24 to EF-2 (Kessler &
Galloway 1982, Prentice & Merrill 1999). Fluorescence spectroscopic studies can be
quite powerful in investigating the interactions between macromolecules. In the current
studies, a covalently linked fluorophore and a FRET-based approach were used to
measure the binding interactions between the two proteins.
FRET involves the transfer of energy from a fluorescent donor in its excited state
to another excitable group, in the acceptor, by a non-radiative dipole-dipole interaction
(Lakowicz, 2000). FRET has been widely employed for assessing the distance between
two loci on macromolecules (e.g., Steer & Merrill 1994; Qin & Sharom 2001). This
technique can also be applied to determine associative interactions since any
phenomenon that affects the donor-acceptor distance will affect the transfer rate, allowing
the phenomenon to be quantified. In the case of macromolecular association
measurements, the determination of a precise donor-acceptor distance is not the main
consideration. Instead, one relies on the simple fact that energy transfer occurs whenever
the donor and acceptors are in close proximity comparable to the Förster distance.
89.
IAEDANS and IAF can alkylate thiol groups, the imidazole moiety of histidine
and the aryl group of tyrosine in proteins (Miki 1990). The reactions of these
fluorophores with thiol group is shown in Figure 24. The absorption spectrum of
AEDANS-PE24 is characterized by absorption maxima at 280 nm and 337 nm (Figure
25A). The labeling stoichiometry of PE24 by IAEDANS can be estimated from the
above data. Fluorescence spectra (Figure 26A) show excitation and emission maxima at
337 and 480 nm, respectively.
Absorption maxima at 280 nm and 490 nm (Figure 25B) characterize the
absorption spectrum of EF-2 labeled with fluorescein. The labeling stoichiometry of EF2 was estimated using this spectrum. The concentration of EF-2 was determined using
the BCA assay as outlined in the methods section (page 46). Fluorescence spectra
(Figure 26B) show excitation and emission maxima at 492 nm and 515 nm, respectively.
The emission spectrum of the AEDANS fluorophore overlaps with the absorption
spectrum of fluorescein, making this combination of fluorophores a good donor-acceptor
pair for FRET studies (Figure 27). Hence, an attempt was made to monitor proteinprotein interactions by measuring fluorescence energy transfer between the AEDANS
moiety in PE24 and the fluorescein fluorophore associated with EF-2. Figure 28 shows
the emission spectra of PE24-AEDANS as a function of labeled EF-2 concentration. The
AEDANS-PE24, when excited at 337 nm shows maximum fluorescence emission at 480
nm. The addition of fluorescein-labeled EF-2 results in an approximately 30% decrease
in fluorescence intensity (Figure 30). This decrease could be due to a change in the local
environment of the probe or to energy transfer from AEDANS present in PE24 to
fluorescein associated with EF-2. The former possibility was assessed by monitoring the
90.
91.
92.
93.
94.
95.
96.
emission spectra of labeled fluorescein-EF-2 excited at 490 nm as a function of
concentration of AEDANS-PE24. A 30% increase of fluorescence intensity at 510 nm
was found to accompany the interaction. Therefore, the decrease of fluorescence at 460
nm (with excitation at 337 nm) noted above could be ascribed to fluorescence energy
transfer between the AEDANS moiety of PE24 and the fluorescein linked to EF-2.
Figure 31 shows a typical isotherm where the increase in the fluorescence
intensity at 460 nm as AF-EF-2 and AEDANS-PE24 concentrations approach a ratio of
1.0 and there is no further change when the two proteins are in 1:1 stoichiometry. These
findings are indicative of a specific interaction between the two proteins.
Having determined the stoichiometry, the same approach was used to obtain
dissociation constants for the protein-protein interaction. All subsequent titrations were
performed by keeping the concentration of the donor constant and varying the
concentration of the acceptor, in order to minimize the overlap (the detection of acceptor
fluorescence at the donor emission wavelength) between the fluorophores and render the
signals more reproducible.
Determination of the ADPRT activity of AEDANS-PE24 derivatives indicated
that the modification had no significant effect on either the interaction with EF-2 or
NAD+, with the catalytic activity being similar to that of parent PE24. Introducing a
fluorescence moiety in EF-2 led to four-fold diminution in the ability to serve as a
substrate when ADPRT activity was performed using the parent PE24.
97.
98.
4.2.6 Influence of NAD+ on EF-2 binding to PE24
The requirement of NAD+ in the binding of the EF-2 to PE24 has been a topic of
debate. ELISA based experiments done in our laboratory and others have indicated that
presence of NAD+ is required for the binding interaction between EF-2 and PE24
(Prentice & Merrill 1999, Kessler & Galloway 1982). On the other hand, Peterson and
coworkers have observed that presence of NAD+ was not required for such an interaction.
These differences in the observations may be attributed to the experimental conditions
involved in the techniques chosen to monitor protein-protein interactions.
In order to acquire detailed valid thermodynamic data to define the binding
interactions between EF-2 and PE24, we looked for a simple and reliable approach that
allows collection of a large amount of accurate data under well-controlled solution
conditions. To this end, the FRET-based assay has been useful in monitoring the
interaction between the two proteins. The results show that the dissociation constant of
PE24.EF-2 is about 2.7 M.
The binding of EF-2 to PE24 was examined both in the absence as well as in the
presence of -TAD. The similarity in binding isotherms was indicative of PE24 of having
no NAD+ requirement for interaction with EF-2. The values for Bmax and Kd derived
from the isotherms are given in Table 8 and show that the binding of EF-2 to PE24 does
not depend on the presence of -TAD.
The interaction of EF-2 with each of the PE24 variants was investigated using the
above-mentioned assay and as the results in Table 9 indicate the binding affinity of all the
variants towards EF-2 was very similar. The above assay is the first report of an
99.
100.
approach, which provides a simple method of monitoring interactions between PE24 and
its substrate EF-2. Since its development other members of our laboratory have utilized
this assay to investigate other structural aspects of the catalytic domain of PE24 such as
the involvement of the catalytic loop in the modulation of ADPRT activity (Yates &
Merrill 2001).
Temperature variation experiments were carried out in order to determine the free
energy associated with the interaction of PE24 and EF-2. The binding affinity of EF-2
was measured as a function of temperature and, using Van’t Hoff’s equation, the enthalpy
of PE24-EF-2 interaction was calculated to be –34  1 kJ.mol-1.
4.2.7 Effect of salt on protein-protein interactions
The final step in purification of EF-2 involves chromatography on Q-Sepharose,
and the EF-2 is usually recovered in the fractions eluted with buffer containing high
concentrations of KCl (300 mM). The ADPRT activity of PE24 has been shown to be
sensitive to the ionic strength of the reaction mixture, with significant inhibition at
moderate salt concentrations (Carrol et al., 1988). As indicated earlier, PE24 has been
found to be capable of its normal catalytic function at concentrations of KCl  100 mM (a
situation that prevails under assay conditions). It has been proposed that the observed
salt effects on the activity of PE24 may be a reflection of the influence of ionic strength
on the electrostatic interactions involved with EF-2.
The structural integrity of EF-2 has been found to be very sensitive to salt
concentrations. The destabilizing influence of salt on EF-2 is further indicated by the
observation that EF-2 in buffer containing KCl (50 mM), upon exposure to –70º C,
101.
loses >50% of its ability to serve as a substrate in the PE24 catalyzed ADP-ribosylation
reaction while similar treatment in the presence of KCl (300 mM) fails to have such
adverse effect (Gerry Prentice, personal communication.
The interaction of EF-2 with PE24 has been studied by employing an enzymelinked immunosorbent assay (ELISA) (Prentice & Merrill 1999). In this study the
interaction of the two proteins was shown to be salt dependent. Although the ELISA
method can be a useful approach, it is an indirect approach requiring the use of several
proteins (PE24, BSA, EF-2, anti-PE24, APase/anti rabbit) a feature, which makes it often
difficult to achieve an unambiguous interpretation. Hence, the FRET-based assay was
used to study the effect of KCl concentration on the interaction between PE24 and EF-2.
The binding of EF-2 to PE24 was assessed as a function of increasing KCl
concentration in the assay. The EF-2 binding to PE24 is not significantly influenced by
increase in KCl concentrations (Figure 31). In contrast, the catalytic activity of PE24
declines steadily with increasing concentrations of KCl in the assay, with almost
complete loss of activity noted at 800 mM. Since enzymatic activity is regained upon
dilution <150 mM, it would appear that ionic strength plays an important role in the
catalytic function of PE24 rather than influencing protein-protein interactions.
102.
103.
4.3
Structure-Activity Studies of inhibitors of ETA
In an attempt to gain insight into the catalytic mechanism involved in the ETA
catalyzed ADPRT reaction, the effect of several compounds that have been shown to
inhibit PARPs was examined. The compound Naph was found to function as a potent
inhibitor of ETA’s ADPRT activity. Naph is the parent compound of many dyes,
fluorescent brighteners, and dye intermediates. These compounds constitute a very
versatile class of compounds that have been used in a large variety of areas. The most
recent application of naphthalenic imides has been in the fields of biology and medicine,
particularly in anticancer therapy (Brana et al., 1980; 1993).
4.3.1 Interaction between PE24 and Naph
The binding of NAD+ to PE24 has been found to be accompanied by the
quenching of the latter’s intrinsic fluorescence and this feature has been exploited to
monitor the interaction (Beattie et. al., 1996). In the current study the same approach was
employed to monitor the interaction of Naph and other compounds with PE24. Figure 32
illustrates the chemical structure of inhibitors used in the current study.
The change in fluorescence intensity of PE24 was recorded as a function of
inhibitor concentration. This spectroscopic titration curve was then converted to an
isotherm for subsequent determination of binding parameters. The results of titration of
PE24 with Naph are shown in Figure 34. The data were consistent with the protein
interacting with the ligand at two distinct sites with KD1 and KD2 values of 54  6 nM and
1.2  0.1 M, respectively. The KD values for Naph and other inhibitors are given in
104.
105.
106.
Table 10. Some of these compounds are analogs of Naph and were found to mimic Naph
by being able to bind at two distinct sites of PE24.
4.3.2 Effect of selected compounds on ADPRT activity of PE24
The influence of Naph and other compounds on the ADPRT activity of PE24 was
examined using the fluorometric assay procedure. The ADPRT activity of PE24 was
determined over a wide range of inhibitor concentration in the assay. The profile of
ADPRT activity versus inhibitor concentration was used to determine the IC50 values.
The IC50 values noted in the case of Naph and other inhibitors are presented in Table 10.
For the determination of the type of inhibition exerted by these compounds, PE24
was incubated with the desired inhibitor for a period of 10 min prior to initiation of the
assay. This preincubation period was selected on the basis of preliminary studies, which
indicated that inhibition of the enzyme reached a maximum value after 5 minutes of
interaction.
In dealing with many of these compounds, we had two technical difficulties; one
was the low solubility of compounds in aqueous medium. This difficulty was overcome
in most cases by the use of an elaborate dilution protocol as outlined in the methods,
which included use of DMSO and ethanol as a vehicle for these compounds. Special
attention was paid to the final concentrations of DMSO and ethanol present in the assay
and the amounts used did not affect the ADPRT activity. The second difficulty was the
fluorescent nature of some of the compounds tested. At low concentrations the
107.
108.
fluorescent properties of these compounds did not interfere with the fluorescence-based
ADPRT assay which meant that only potent inhibitors could be measured accurately.
A comparison of potency of these compounds and their structure indicated that a
common structural feature shared by all very strong inhibitors was a carboxamide
attached to an aromatic ring or a carbamoyl group built in a poly aromatic heterocyclic
skeleton to form a fused aromatic lactam or imide. The IC50 values of the most potent
compounds ranged from 87 nm to 1.5 M.
It is also important to note that these inhibitors are isosteric to the nicotinamide
moiety of NAD+. It seems that potency of Naph arises from the fact that its amide group
is fixed in a hetero ring. The presence of an amino group at the 4 position of Naph
seemed to alter its inhibitory effect, although not dramatically, perhaps due to steric
interactions with the NAD binding pocket of the toxin.
The initial rates of ADPRT activity of PE24 samples exposed to Naph were
determined as a function of NAD+ concentration. The inhibition of ETA by Naph
appeared to be of a complex nature. The double reciprocal plot of the data (Figure 34)
indicated that Naph functions as a competitive inhibitor when its concentration was kept
below 300 nM. However, at higher concentrations this model was no longer supported
by the data. As mentioned before this compound was not soluble in aqueous medium and
it is conceivable that at concentrations above 300 nM the dynamic precipitation of Naph
interferes with the assay hence resulting in complex data. On the other hand, it is
109.
110.
possible that Naph binds the second binding site on the enzyme and therefore causes a
more complex inhibition profile. However, we think that the main reason for this
behavior stems from the solubility issues of Naph.
4.3.3
Analysis of reversibility of inhibition of PE24 by 1,8-naphthalimide
Experiments were performed to determine the reversibility of the inhibition
phenomenon. Extensive dialysis of PE24 was found to result in an approximately 50%
loss of its ADPRT activity indicating the labile nature of the protein under the
experimental conditions. PE24 sample treated with Naph was found to lose 80% of its
ADPRT activity. Upon dialysis as above, the sample was found to regain its activity
(approximately 42% of the expected value) and when compared to the results in the
control this finding constitutes a 90% recovery in enzymatic activity (Table 11).
The results indicate that inhibition of ADPRT activity by 1,8-naphthalimide is
reversible and non-covalent in nature. These findings were further confirmed using mass
spectrometry, which showed no change in mass of protein upon its incubation with Naph
(Figure 36). The mass spectrum of the PE24 with 1,8-naphthalimide showed the
presence of a peak representing a mass of starting material PE24 (24532 kDa). The
spectrum (Figure 36A) shows the presence of very small contaminating peaks and none
of these peaks correspond to that of PE24 covalently modified by Naph.
The decrease in fluorescence accompanying the interaction of PE24 with Naph
and other mentioned compounds suggested that the inhibitors may be binding in the
vicinity of tryptophan residues present in the protein. There are three tryptophan residues
111.
112.
113.
114.
in the NAD+ binding pocket of PE24. NMR studies (Figure 37) failed to show any
covalent interaction between N-acetyl tryptophanamide (NATA), the model compound
for tryptophan, and Naph. However, the ability of Naph to quench the fluorescence of
NATA as efficiently as noted in the case of tryptophan residues present in PE24 suggests
that the inhibitor binds near the NAD+ binding pocket of PE24.
The kinetic data presented indicated that Naph binds the NAD+ binding site of the
enzyme and inhibits the ADPRT reaction reversibly. Similar compounds known to
inhibit PARP activity have been co-crystallized with the catalytic domain of various
PARPs and have been shown to bind this enzyme in the NAD+ binding site. The binding
of Naph to PE24 was further investigated using molecular modeling studies.
The structure of Naph was generated using Sybyl 6.7, energy minimized using the
Tripos forcefield and superimposed on that of the nicotinamide moiety in the x-ray
crystal structure of ETA bound to hydrolysis products of NAD+ (Li et al., 1995) as shown
in Figure 37. In superimposing the inhibitor onto the structure of nicotinamide, the
structural feature common to both compounds, namely, the aromatic ring and
carboxamide moiety atoms were used.
Completion of the docking of 1,8-naphthalimide involved deletion of the
nicotinamide structure from the protein crystallographic file and merging the structure of
1,8-naphthalimide into the protein structure. The geometry of this hypothetical complex
was optimized in a fashion similar to that outlined previously. In the present study the
computation employed the Tripos force field which has been shown to be relatively
reliable for small organic molecules and peptides (Homans, 1990) and has been used
successfully in the analysis of a variety of enzyme-inhibitor complexes (Clark et al.,
115.
1988). Electrostatic interactions were included based on partial atomic charges assigned
using the Gasteiger-Huckel method (Gasteiger et al., 1980; Purcel et al., 1967).
In the model, which emerged from this analysis, the inhibitor was observed to
have favorable contacts with amino acids comprising the NAD binding site. Distances
between atoms of 1,8-naphthalimide in the annealed complex described above and of the
amino acid residues of the binding site are in indicated in Figure 38. A residue is shown
only if atoms of that residue are  3.7 Å from the inhibitor atom. This model compares
favourably to the structure of PARP inhibitor complexes reported by Ruf and coworkers
(Ruf et al., 1999). These studies suggest that these compounds mimic the nicotinamide
portion of NAD+ in the binding site.
The structure of Naph in the binding site of ETA is in close proximity to Glu 553
(essential catalytic residue). This residue is thought to be involved in stabilization of the
oxocarbenium-like ion in the transition state proposed for ADPRT reaction. It is feasible
to suggest that Naph exerts its inhibitory action by mimicking the transition state. Most
of the residues involved in the binding of NAD+ are shown to be in favorable interactions
with Naph in the model studies.
The docking experiment is a crude model of the enzyme.inhibitor interactions
since we did not account for unknown structural changes as a consequence binding of
inhibitor and the second substrate EF-2. However, with the Naph structure fitted in the
active site, the emerging model gives further insights into the features of the catalytic site
that might suggest strategies for designing transition state inhibitors of ETA.
116.
117.
Discussion
The goals of this study were to gather insight into the kinetic mechanism of the
ADPRT reaction, illustrating the validity of the fluorescence-based assay, and to
characterize the kinetic parameters of the toxin-catalyzed reaction for both substrates.
The results herein are the first report of such parameters for the EF-2 substrate of ETA.
These results demonstrate the suitability of the fluorometric assay for monitoring the
ADP-ribosylation reaction catalyzed by PE24. The kinetic parameters from this method
are very similar to those recorded using a traditional radioactive substrate assay in our
laboratory. Current assays for ADP-ribosylation are limited because of their
discontinuous nature, requiring the laborious process of individually processing
numerous samples, and result in the assembly of only a partial reaction progress curve.
Previously, the use of the traditional radioactive assays for ADP-ribosylation
made it difficult to study the inhibition mechanisms of specific inhibitors for ETA
catalyzed ADP-ribosylation reactions. However, the fluorometric procedure is more
rapid, sensitive, and has advantage of providing continuous acquisition of data.
Furthermore, the spectrofluorometric ADP-ribosylation assay described herein is
amenable to automation in assay procedures involving the use of a fluorescence
microplate reader. This makes it ideal as a rapid screening assay for the detection of
enzyme inhibitor candidates.
This assay utilizes the fluorescence property of the NAD+ analogue, -NAD+,
(Figure 39) which is due to the etheno bridge between C-1 and N6 positions of the
adenine ring (Klebl et al., 1996). This analogue has been shown to be interchangeable
118.
119.
with NAD+ in binding with several dehydrogenases (Barrio et al., 1972). The
fluorescence intensity (quantum yield) of -NAD+ is less than that for the fluorescent
product of -NAD+ hydrolysis, -AMP, most likely as a result of intramolecular
fluorescence quenching by the nicotinamide ring (Barrio et al., 1972).
Gruber and Leonard (Gruber et al., 1975) report that the quantum yield of NAD+ in aqueous buffered medium is 0.028, and its fluorescence lifetime is short, 2.1 ns.
This contrasts sharply with the reported quantum yield value of 0.56 for 5-AMP (Gruber
et al., 1975). Therefore, the reaction can be followed spectroscopically by monitoring the
increase in fluorescence intensity, which results from the enzymatic cleavage of the 1,N6
glycosidic bond of -NAD+. This continuous kinetic assay enabled the collection of
approximately 600 data points (20pts/sec), which greatly enhanced the statistical
precision of the measured kinetic parameters for the ADPRT reaction.
The use of fluorescent NAD+ derivatives has proven to be advantageous in other
systems as well, since it provides a more direct method for kinetic studies. Basso and coworkers (Basso et. al., 1997) have used -NAD+ to study the kinetics of binding for
glutamate dehydrogenase. They were able to probe the nucleotide binary and ternary
complex formation using -NAD+. Davis and co-workers (Davis et. al., 1998) have also
shown the high sensitivity of fluorescent derivatives of NAD+ in measuring ADPribosylation reactions. They used -NAD+ to detect PARP activity in an
immunohistochemical assay in which a specific antibody to etheno adenosine was used to
detect the production of modified poly-ADP-ribose in cell extracts.
Other investigators, including some in our own laboratory, have observed that the
ADPRT activity of the diphthamide-specific ribosyltransferases is sensitive to the ionic
120.
strength of the reaction mixture (Prentice et. al., 1999, Collier et. al., 1988).
Furthermore, at moderate salt concentrations, total loss of ADPRT activity for the
enzyme is observed. In the present study, it was seen that at concentrations above 500
mM KCl there was almost complete inhibition of activity as indicated in Figure xx. In
order to further pursue the mechanism behind these observations, the effect of KCl
concentration on NAD+ binding capacity of PE24 was also explored. Thus, the high salt
sensitivity of the toxin’s ADPRT activity suggests that a major component of the toxin’s
interaction with EF-2 must involve an electrostatic effect.
The utility of the method is indicated by its applicability over a wide range of pH,
temperature and ionic strength values. The kinetic data in this report show that PE24, the
C-terminal fragment of ETA, is an efficient catalyst like its parent protein in promoting
the ADP-ribosylation reaction. Interestingly, the catalytic function of PE24 at 37C
remains relatively unimpaired, indicating the preservation of its active conformation
under conditions of the assay.
The effect of pH on the ADPRT activity of PE24 was explored in order to identify
changes in the ionization state of amino acid side chains, which are essential for the
catalytic activity of the enzyme. Here, we report a detailed analysis of the ionization
involved, where values of both the KM and kcat parameters were obtained over a pH range
of 2-11 (page 69). The curve in Figure 17A for PE24 reveals the presence of titratable
groups that affect the ADPRT activity of the enzyme. However, the assignment of pKa
values for the catalytic residues is much more complex, since one of the substrates in this
enzyme-catalyzed reaction is also a protein with numerous potentially ionizable groups,
namely, EF-2.
121.
Due to an inherent difficulty in experiments with EF-2 as the limiting substrate,
pH experiments were not conducted for the determination of the apparent kinetic
parameters for the EF-2 substrate. A plot of kcat/KM against pH illustrates that two pK
values could describe the pH profile for PE24. Curiously, this plot for the pHdependence of PE24 is considerably narrower than the same plot for PE40, or for the
whole toxin (Beattie et. al., 1996). As illustrated in Figure 17, the pH profile of kcat is
broader than the kcat/KM profile, which is indicative of one or more His residue(s)
involved in the catalytic process. A plot of kcat against pH was narrower than the
corresponding kcat/KM plot, which is indicative of greater cooperativity of the ionization
process for the PE24-NAD+ complex. The acidic and alkaline pK values calculated for
this complex were 7.0 and 8.9, respectively, which also suggests a His residue involved
in the interaction of substrate with the enzyme.
Alternatively, since there are no cysteines in PE24, the alkaline value could also
be attributed to a Tyr residue, such as Tyr 481, found in the nicotinamide-binding pocket.
These findings complement the mutational experiments that indicate His residues 426
and 440 (Prentice et al., 2001) are essential for activity, and are located near the active
site cleft where they may interact with substrate. However, caution must be exercised
when interpreting these pH plots and inferring potential catalytic residues, since
environmentally shifted pKas for ionizable active site residues in enzymes have been well
documented (Fersht, 1999).
The activity of PE24 declines steeply at temperatures above 30 C, which is
indicative of thermal denaturation of the enzyme. This finding was further confirmed by
differential scanning calorimetry experiments. The catalytic fragment, PE24, had a
122.
melting temperature of 31C. The objective of the studies presented here was to use a
smaller catalytic fragment, namely, PE24. The data indicated that there were no
significant changes in the steady state kinetic parameters of the truncated toxin with
respect to the NAD+ substrate, as compared to the whole toxin. Although the fragment
exhibits a lower thermal stability, it still possesses a significant level of activity at 37 C.
It is not surprising that PE24 is unstable at temperatures below physiological
conditions. PE24 does not contain the domain II portion of the toxin, which is part of the
in vivo biologically active 37 kDa toxin fragment that is produced upon infection of the
host cell by ETA. This may account for the differences in the relative folded stability of
PE24 and the biologically active peptide. Nonetheless, it is of interest to note that PE24,
when expressed in Saccharomyces cerevisiae grown at 30 C, is extremely toxic to the
yeast host cells and that an active site mutant, E553D, is much less cytotoxic (C.
Thompson, MSc. Thesis 1999). The thermal effect on PE24 ADPRT activity indicates
that this catalytic fragment (domain III) is less stable than either PE40 (part of domain II
and all of domain III), or the whole toxin. Urea denaturation experiments have also
demonstrated that the whole toxin and PE40 are more stable than PE24 (Beattie et al.,
1996 & 1999).
Furthermore, the relatively high thermal stability of EF-2 indicates that
the loss of ADPRT activity at temperatures above 37 C is likely due to the thermal
instability of PE24 and not EF-2. It was determined from our earlier folding experiments
involving the whole toxin, PE40, and PE24 (Beattie et al., 1996 & 1999) that PE24
refolds extremely quickly compared to the whole toxin and PE40. The biological
implications of this rapid refolding process of the catalytic domain can perhaps be
explained by the in vivo refolding requirement after translocation to the cytoplasm upon
123.
crossing the endoplasmic reticulum membrane in order to avoid destruction by host
endogenous proteases or other scavenging cellular machinery during the intoxication
process.
In summary, this assay is much more amenable to an automated procedure, which
could provide a high throughput approach to study the enzyme kinetics and catalytic
mechanism of diphthamide-specific ADPRT enzymes such as ETA and diphtheria toxins
as well as NADase and glycohydrolase enzymes. In this study, the improved approach for
the acquisition of the kinetic properties of the toxin ADPRT activity provided additional
insight into the catalytic mechanism and properties of this toxin-enzyme.
The interaction of ETA with one of its substrates, NAD+, has been well
characterized, owing a great part to the availability of the crystal structure of ETA in
complex with NAD+ as well as other biochemical approaches, such as mutational and
affinity labeling studies. On the other hand, the lack of a structure for EF-2 and its overexpression system has rendered the study of ETA interaction with EF-2 very difficult.
One of the objectives of the current study was to develop a direct and easy method of
monitoring interactions between these two proteins. It was anticipated that a direct
method would allow for a conclusive determination of factors effecting this interaction,
and for the identification of residues that might be important in the catalytic activity due
to association with EF-2.
The FRET-based assay described in this thesis is the first record of a direct assay,
which monitors the interaction between PE24 and its substrate EF-2. The quenching of
fluorescence of the AEDANS-PE24 upon addition of the AF-EF-2 is indicative of energy
transfer, which results from interaction between the AEDANS and Fluoresein as a
124.
consequence of binding between PE24 and EF-2. Using FRET, the dissociation constant
of the PE24.EF-2 complex was determined to be 2.7 M ± 0.4. The formation of the
complex between PE24 and EF-2 does not appear to require the presence of the second
substrate NAD+ as previously reported (Kessler et al., 1988). The binding between the
two proteins is not affected by increasing salt concentrations to a significant level.
However, the enzyme activity is significantly disrupted at high salt concentrations. These
findings suggest that high salt concentrations render the enzyme inactive, not by affecting
binding, but rather by influencing the ionic environment of the catalytic residues.
Inhibition of target enzymes with compounds that are specific and show tightbinding characteristics has traditionally been a successful approach in drug discovery.
Using this approach, one can gain insight into the role of binding in catalysis or
molecular recognition. Therefore, the objective of the current study was to employ an
inhibitor-based approach to study the catalytic mechanism of ETA. To this end, we
tested a group of compounds with different inhibitory effects against PE24 activity and
characterized the mode of action of Naph, the most potent of these compounds, in detail.
In this study, we made the following key observations. (i) Naph inhibits ADPRT
activity of PE24 at nM concentrations. (ii) This inhibition is reversible and exhaustive
dialysis of the complex leads to regain of the activity. (iii) At low concentrations of
Naph, the kinetics of the reaction follow a competitive model of inhibition. (iv) The
molecular modeling experiments suggest that Naph binds the enzyme in the substratebinding pocket. Taken together, these findings suggest that Naph may behave as a
transition state analog inhibitor.
125.
The ADPRT reaction mechanism is thought to proceed through an SN1
mechanism. This conclusion concerning the mechanism is based on studies of
substituent effects on the hydrolysis of 2’-substituted nicotinamide arabinosides (Handlon
& Oppenheimer 1991). The mechanism involves the formation of an oxocarbocationic
intermediate. However, structural data obtained by NMR spectroscopy has shown
inversion of configuration for the glycosidic bond of ribosyl diphthamide (Oppenheimer
& Bodley, 1981). This observation can be explained by the notion of restriction in the
accessibility of the C-1 of the nicotinamide ribose to nucelophilic attack by the N-1 of the
imidazole ring of diphthamide, resulting in a backside attack on the oxocabonium ion.
The binding assay and molecular modeling studies suggested that Naph might
bind the NAD binding pocket. The model structure indicates that Naph makes multiple
contacts with residues Tyr 471 and Tyr 480. On the basis of this model, in the inhibitor
complex there is potential for H-bonding interactions between NH of Naph and Gly441
as well as carbonyl oxygen of Naph and His 440. The potency of the inhibitor seems to
be associated with having the amide group fixed in a hetroring. Like NAD, Naph
interacts with residues of the catalytic domain that are generally conserved in the ADPRT
family comprising poly and mono ADPRT.
These observed enzyme-inhibitor interactions in the PE24-Naph complex are
relevant to our understanding of the detail of the chemical mechanism. Particularly
important interactions are those involving the Glu 553 residue, which has been implicated
in stabilizing the transition state structure. In the inhibitor-enzyme complex, this
carboxylate group is at a distance of 3.6 Å from the C9 atom of Naph that is structurally
equivalent to the anomeric carbon atom of the nicotinamide ribose of NAD+. We
126.
propose that Naph acts similar to a TS analogue because it binds tightly yet reversibly to
PE24, and has close structural homology with the proposed ADPRT reaction mechanism
TS.
Classical transition state theory, which proposes that the enzymatic rate
enhancement (Fersht, 1999) is principally due to stabilization of an otherwise unlikely
intermediate, would predict that these TS-like inhibitors resemble the activated substrates
in the TS as closely as chemically possible. Although the ADPRT catalytic rate
enhancement is unknown, the high affinity of Naph relative to substrate implies that there
exists a substrate-like bound state for which the Gibbs free energy is very much lower
than the enzyme-substrate complex.
127.
References
Aevarsson A, Brazhnikov E, Garber M, Zheltonosova J, Chirgadze Y, al Karadaghi S,
Svensson LA, Liljas A. 1994. Three-dimensional structure of the ribosomal translocase:
elongation factor G from Thermus thermophilus. EMBO J. 13:3669-77
Agrawal RK, Penczek P, Grassucci RA, Frank J. 1998. Visualization of elongation factor
G on the Escherichia coli 70S ribosome: the mechanism of translocation. Proc. Natl.
Acad. Sci. U. S. A 95:6134-8
Allured VS, Collier RJ, Carroll SF, McKay DB. 1986. Structure of exotoxin A of
Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc. Natl. Acad. Sci. U. S. A
83:1320-4
Alvarez-Gonzalez R, Althaus FR. 1989. Poly(ADP-ribose) catabolism in mammalian
cells exposed to DNA-damaging agents. Mutat. Res. 218:67-74
Ame JC, Apiou F, Jacobson EL, Jacobson MK. 1999. Assignment of the poly(ADPribose) glycohydrolase gene (PARG) to human chromosome 10q11.23 and mouse
chromosome 14B by in situ hybridization. Cytogenet. Cell Genet. 85:269-70
Ame JC, Rolli V, Schreiber V, Niedergang C, Apiou F, Decker P, Muller S, Hoger T,
Menissier-de Murcia J, de Murcia G. 1999. PARP-2, A novel mammalian DNA damagedependent poly(ADP-ribose) polymerase. J. Biol. Chem. 274:17860-8
Ame JC, Jacobson EL, Jacobson MK. 1999. Molecular heterogeneity and regulation of
poly(ADP-ribose) glycohydrolase. Mol. Cell Biochem. 193:75-81
128.
Armstrong S, Merrill AR. 2001. Application of a fluorometric assay for characterization
of the catalytic competency of a domain III fragment of Pseudomonas aeruginosa
exotoxin A. Anal. Biochem. 292:26-33
Babiychuk E, Cottrill PB, Storozhenko S, Fuangthong M, Chen Y, O'Farrell MK, Van
Montagu M, Inze D, Kushnir S. 1998. Higher plants possess two structurally different
poly(ADP-ribose) polymerases. Plant J. 15:635-45
Barrio JR, Secrist JA, III, Leonard NJ. 1972. A fluorescent analog of nicotinamide
adenine dinucleotide. Proc. Natl. Acad. Sci. U. S. A 69:2039-42
Barrio JR, Secrist JA, III, Leonard NJ. 1972. Fluorescent adenosine and cytidine
derivatives. Biochem. Biophys. Res. Commun. 46:597-604
Basso LA, Engel PC, Walmsley AR. 1997. Kinetic studies on the binding of 1,N6etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum. Biochim.
Biophys. Acta 1340:63-71
Beattie BK, Merrill AR. 1996. In vitro enzyme activation and folded stability of
Pseudomonas aeruginosa exotoxin A and its C-terminal peptide. Biochemistry 35:904251
Beattie BK, Merrill AR. 1999. A fluorescence investigation of the active site of
Pseudomonas aeruginosa exotoxin A. J. Biol. Chem. 274:15646-54
Bell CE, Eisenberg D. 1996. Crystal structure of diphtheria toxin bound to nicotinamide
adenine dinucleotide. Biochemistry 35:1137-49
129.
Berghammer H, Ebner M, Marksteiner R, Auer B. 1999. pADPRT-2: a novel mammalian
polymerizing(ADP-ribosyl)transferase gene related to truncated pADPRT homologues in
plants and Caenorhabditis elegans. FEBS Lett. 449:259-63
Bernofsky C. 1980. Physiology aspects of pyridine nucleotide regulation in mammals.
Mol. Cell Biochem. 33:135-43
Brana MF, Castellano JM, Roldan CM, Santos A, Vazquez D, Jimenez A. 1980.
Synthesis and mode(s) of action of a new series of imide derivatives of 3-nitro-1,8
naphthalic acid. Cancer Chemother. Pharmacol. 4:61-6
Brana MF, Castellano JM, Moran M, Perez d, V, Romerdahl CR, Qian XD, Bousquet P,
Emling F, Schlick E, Keilhauer G. 1993. Bis-naphthalimides: a new class of antitumor
agents. Anticancer Drug Des 8:257-68
Brandhuber BJ, Allured VS, Falbel TG, McKay DB. 1988. Mapping the enzymatic active
site of Pseudomonas aeruginosa exotoxin A. Proteins 3:146-54
Cammarano P, Creti R, Sanangelantoni AM, Palm P. 1999. The archaea monophyly
issue: A phylogeny of translational elongation factor G(2) sequences inferred from an
optimized selection of alignment positions. J. Mol. Evol. 49:524-37
Carroll SF, Collier RJ. 1987. Active site of Pseudomonas aeruginosa exotoxin A.
Glutamic acid 553 is photolabeled by NAD and shows functional homology with
glutamic acid 148 of diphtheria toxin. J. Biol. Chem. 262:8707-11
Carroll SF, Collier RJ. 1988. Amino acid sequence homology between the enzymic
domains of diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Mol. Microbiol.
2:293-6
130.
Carroll SF, Barbieri JT, Collier RJ. 1988. Diphtheria toxin: purification and properties.
Methods Enzymol. 165:68-76
Carroll SF, Collier RJ. 1988. Diphtheria toxin: quantification and assay. Methods
Enzymol. 165:218-25
Carson DA, Seto S, Wasson DB, Carrera CJ. 1986. DNA strand breaks, NAD
metabolism, and programmed cell death. Exp. Cell Res. 164:273-81
Cassel D, Pfeuffer T. 1978. Mechanism of cholera toxin action: covalent modification of
the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc. Natl. Acad.
Sci. U. S. A 75:2669-73
Cervantes-Laurean D, Minter DE, Jacobson EL, Jacobson MK. 1993. Protein glycation
by ADP-ribose: studies of model conjugates. Biochemistry 32:1528-34
Cervantes-Laurean D, Jacobson EL, Jacobson MK. 1996. Glycation and glycoxidation of
histones by ADP-ribose. J. Biol. Chem. 271:10461-9
Chaudhary VK, Jinno Y, FitzGerald D, Pastan I. 1990. Pseudomonas exotoxin contains a
specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl.
Acad. Sci. U. S. A 87:308-12
Clapper DL, Walseth TF, Dargie PJ, Lee HC. 1987. Pyridine nucleotide metabolites
stimulate calcium release from sea urchin egg microsomes desensitized to inositol
trisphosphate. J. Biol. Chem. 262:9561-8
Cohen SN, Chang AC, Hsu L. 1972. Nonchromosomal antibiotic resistance in bacteria:
genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U. S.
A 69:2110-4
131.
Collier RJ. 1975. Diphtheria toxin: mode of action and structure. Bacteriol. Rev. 39:54-85
Cryz SJ, Jr., Friedman RL, Iglewski BH. 1980. Isolation and characterization of a
Pseudomonas aeruginosa mutant producing a nontoxic, immunologically crossreactive
toxin A protein. Proc. Natl. Acad. Sci. U. S. A 77:7199-203
Czworkowski J, Wang J, Steitz TA, Moore PB. 1994. The crystal structure of elongation
factor G complexed with GDP, at 2.7 A resolution. EMBO J. 13:3661-8
D'Amours D, Desnoyers S, D'Silva I, Poirier GG. 1999. Poly(ADP-ribosyl)ation
reactions in the regulation of nuclear functions. Biochem. J. 342 ( Pt 2):249-68
Domenighini M, Montecucco C, Ripka WC, Rappuoli R. 1991. Computer modelling of
the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of
NAD binding. Mol. Microbiol. 5:23-31
Domenighini M, Magagnoli C, Pizza M, Rappuoli R. 1994. Common features of the
NAD-binding and catalytic site of ADP- ribosylating toxins. Mol. Microbiol. 14:41-50
Durkacz BW, Omidiji O, Gray DA, Shall S. 1980. (ADP-ribose)n participates in DNA
excision repair. Nature 283:593-6
Elzaim HS, Chopra AK, Peterson JW, Goodheart R, Heggers JP. 1998. Generation of
neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa
exotoxin A. Infect. Immun. 66:2170-9
Faraone-Mennella MR, Gambacorta A, Nicolaus B, Farina B. 1998. Purification and
biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the
thermophilic archaeon Sulfolobus solfataricus. Biochem. J. 335 ( Pt 2):441-7
132.
FitzGerald D, Morris RE, Saelinger CB. 1980. Receptor-mediated internalization of
Pseudomonas toxin by mouse fibroblasts. Cell 21:867-73
Fujimura S, Hasegawa S, Shimizu Y, Sugimura T. 1967. Polymerization of the adenosine
5'-diphosphate-ribose moiety of nicotinamide-adenine dinucleotide by nuclear enzyme. I.
Enzymatic reactions. Biochim. Biophys. Acta 145:247-59
Gomez-Lorenzo MG, Spahn CM, Agrawal RK, Grassucci RA, Penczek P, Chakraburtty
K, Ballesta JP, Lavandera JL, Garcia-Bustos JF, Frank J. 2000. Three-dimensional cryoelectron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome
at 17.5 A resolution. EMBO J. 19:2710-8
Goodwin PM, Lewis PJ, Davies MI, Skidmore CJ, Shall S. 1978. The effect of gamma
radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells.
Biochim. Biophys. Acta 543:576-82
Graeff RM, Walseth TF, Fryxell K, Branton WD, Lee HC. 1994. Enzymatic synthesis
and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with
ADP-ribosyl cyclase activity. J. Biol. Chem. 269:30260-7
Gray GL, Smith DH, Baldridge JS, Harkins RN, Vasil ML, Chen EY, Heyneker HL.
1984. Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A
structural gene of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A 81:2645-9
Gruber BA, Leonard NJ. 1975. Dynamic and static quenching of 1,N6-ethenoadenine
fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3(indol-3-yl) propyl) adenine. Proc. Natl. Acad. Sci. U. S. A 72:3966-9
Hamood AN, Wick MJ, Iglewski BH. 1990. Secretion of toxin A from Pseudomonas
aeruginosa PAO1, PAK, and PA103 by Escherichia coli. Infect. Immun. 58:1133-40
133.
Hashimoto T, Hasegawa M. 1996. Origin and early evolution of eukaryotes inferred from
the amino acid sequences of translation elongation factors 1alpha/Tu and 2/G. Adv.
Biophys. 32:73-120
Herrero-Yraola A, Bakhit SM, Franke P, Weise C, Schweiger M, Jorcke D, Ziegler M.
2001. Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in
mitochondria. EMBO J. 20:2404-12
Hillyard D, Rechsteiner M, Manlapaz-Ramos P, Imperial JS, Cruz LJ, Olivera BM. 1981.
The pyridine nucleotide cycle. Studies in Escherichia coli and the human cell line
D98/AH2. J. Biol. Chem. 256:8491-7
Homans SW. 1990. A molecular mechanical force field for the conformational analysis
of oligosaccharides: comparison of theoretical and crystal structures of Man alpha 13Man beta 1-4GlcNAc. Biochemistry 29:9110-8
Honjo T, Nishizuka Y, Hayaishi O. 1968. Diphtheria toxin-dependent adenosine
diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J.
Biol. Chem. 243:3553-5
Hudson EN, Weber G. 1973. Synthesis and characterization of two fluorescent sulfhydryl
reagents. Biochemistry 12:4154-61
Hwang J, Fitzgerald DJ, Adhya S, Pastan I. 1987. Functional domains of Pseudomonas
exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 48:129-36
Iglewski BH, Sadoff JC. 1979. Toxin inhibitors of protein synthesis: production,
purification, and assay of Pseudomonas aeruginosa toxin A. Methods Enzymol. 60:780-93
134.
Jacobson EL, Lange RA, Jacobson MK. 1979. Pyridine nucleotide synthesis in 3T3 cells.
J. Cell Physiol 99:417-25
Jinno Y, Chaudhary VK, Kondo T, Adhya S, Fitzgerald DJ, Pastan I. 1988. Mutational
analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas
exotoxin which reduce cell binding and animal toxicity. J. Biol. Chem. 263:13203-7
Kessler SP, Galloway DR. 1992. Pseudomonas aeruginosa exotoxin A interaction with
eucaryotic elongation factor 2. Role of the His426 residue. J. Biol. Chem. 267:19107-11
Klebl BM, Pette D. 1996. A fluorometric assay for measurement of mono-ADPribosyltransferase activity. Anal. Biochem. 239:145-52
Kounnas MZ, Morris RE, Thompson MR, Fitzgerald DJ, Strickland DK, Saelinger CB.
1992. The alpha 2-macroglobulin receptor/low density lipoprotein receptor- related
protein binds and internalizes Pseudomonas exotoxin A. J. Biol. Chem. 267:12420-3
Kunkel TA. 1985. Rapid and efficient site-specific mutagenesis without phenotypic
selection. Proc. Natl. Acad. Sci. U. S. A 82:488-92
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227:680-5
Laemmli UK, Beguin F, Gujer-Kellenberger G. 1970. A factor preventing the major head
protein of bacteriophage T4 from random aggregation. J. Mol. Biol. 47:69-85
Lee HC. 1997. Mechanisms of calcium signaling by cyclic ADP-ribose and NAADP.
Physiol Rev. 77:1133-64
135.
Lee HC, Aarhus R. 1997. Structural determinants of nicotinic acid adenine dinucleotide
phosphate important for its calcium-mobilizing activity. J. Biol. Chem. 272:20378-83
Lee HC. 1997. Mechanisms of calcium signaling by cyclic ADP-ribose and NAADP.
Physiol Rev. 77:1133-64
Lee HC, Aarhus R. 1997. Structural determinants of nicotinic acid adenine dinucleotide
phosphate important for its calcium-mobilizing activity. J. Biol. Chem. 272:20378-83
Leppla SH, Martin OC, Muehl LA. 1978. The exotoxin P. aeruginosa: a proenzyme
having an unusual mode of activation. Biochem. Biophys. Res. Commun. 81:532-8
Li M, Dyda F, Benhar I, Pastan I, Davies DR. 1995. The crystal structure of
Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP:
conformational differences with the intact exotoxin. Proc. Natl. Acad. Sci. U. S. A
92:9308-12
Li M, Dyda F, Benhar I, Pastan I, Davies DR. 1996. Crystal structure of the catalytic
domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide
analog: implications for the activation process and for ADP ribosylation. Proc. Natl.
Acad. Sci. U. S. A 93:6902-6
Lindahl T, Satoh MS, Poirier GG, Klungland A. 1995. Post-translational modification of
poly(ADP-ribose) polymerase induced by DNA strand breaks. Trends Biochem. Sci.
20:405-11
Liu PV. 1966. The roles of various fractions of Pseudomonas aeruginosa in its
pathogenesis. 3. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis.
116:481-9
136.
Lukac M, Collier RJ. 1988. Pseudomonas aeruginosa exotoxin A: effects of mutating
tyrosine-470 and tyrosine-481 to phenylalanine. Biochemistry 27:7629-32
Miki M. 1990. Resonance energy transfer between points in a reconstituted skeletal
muscle thin filament. A conformational change of the thin filament in response to a
change in Ca2+ concentration. Eur. J. Biochem. 187:155-62
Moss J, Vaughan M. 1977. Mechanism of action of choleragen. Evidence for ADPribosyltransferase activity with arginine as an acceptor. J. Biol. Chem. 252:2455-7
Moss J, Jacobson MK, Stanley SJ. 1985. Reversibility of arginine-specific mono(ADPribosyl)ation: identification in erythrocytes of an ADP-ribose-L-arginine cleavage
enzyme. Proc. Natl. Acad. Sci. U. S. A 82:5603-7
Moss J, Vaughan M. 1988. ADP-ribosylation of guanyl nucleotide-binding regulatory
proteins by bacterial toxins. Adv. Enzymol. Relat Areas Mol. Biol. 61:303-79
Nakagawara K, Mori M, Takasawa S, Nata K, Takamura T, Berlova A, Tohgo A,
Karasawa T, Yonekura H, Takeuchi T, . 1995. Assignment of CD38, the gene encoding
human leukocyte antigen CD38 (ADP- ribosyl cyclase/cyclic ADP-ribose hydrolase), to
chromosome 4p15. Cytogenet. Cell Genet. 69:38-9
Nishizuka Y, Ueda K, Nakazawa K, Hayaishi O. 1967. Studies on the polymer of
adenosine diphosphate ribose. I. Enzymic formation from nicotinamide adenine
dinuclotide in mammalian nuclei. J. Biol. Chem. 242:3164-71
Nissen P, Kjeldgaard M, Thirup S, Polekhina G, Reshetnikova L, Clark BF, Nyborg J.
1995. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP
analog. Science 270:1464-72
137.
Oei SL, Griesenbeck J, Schweiger M. 1997. The role of poly(ADP-ribosyl)ation. Rev.
Physiol Biochem. Pharmacol. 131:127-73
Ogata M, Chaudhary VK, Pastan I, Fitzgerald DJ. 1990. Processing of Pseudomonas
exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment
that is translocated to the cytosol. J. Biol. Chem. 265:20678-85
Okazaki IJ, Moss J. 1998. Glycosylphosphatidylinositol-anchored and secretory isoforms
of mono- ADP-ribosyltransferases. J. Biol. Chem. 273:23617-20
Papini E, Santucci A, Schiavo G, Domenighini M, Neri P, Rappuoli R, Montecucco C.
1991. Tyrosine 65 is photolabeled by 8-azidoadenine and 8-azidoadenosine at the NAD
binding site of diphtheria toxin. J. Biol. Chem. 266:2494-8
Perentesis JP, Phan LD, Gleason WB, LaPorte DC, Livingston DM, Bodley JW. 1992.
Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of
expression, and G-domain modeling. J. Biol. Chem. 267:1190-7
Perentesis JP, Miller SP, Bodley JW. 1992. Protein toxin inhibitors of protein synthesis.
Biofactors 3:173-84
Prentice GA, Merrill AR. 1999. An enzyme-linked immunosorbent assay for the
association of the catalytic domain of diphthamide-specific ribosyltransferases to
eukaryotic elongation factor-2. Anal. Biochem. 272:216-23
Purnell MR, Whish WJ. 1980. The effect of phenones on poly(adenosine diphosphate
ribose) synthetase from porcine thymus [proceedings]. Biochem. Soc. Trans. 8:175-6
Qu Q, Sharom FJ. 2001. FRET analysis indicates that the two ATPase active sites of the
P- glycoprotein multidrug transporter are closely associated. Biochemistry 40:1413-22
138.
Riddles PW, Blakeley RL, Zerner B. 1979. Ellman's reagent: 5,5'-dithiobis(2nitrobenzoic acid)--a reexamination. Anal. Biochem. 94:75-81
Ruf A, Rolli V, de Murcia G, Schulz GE. 1998. The mechanism of the elongation and
branching reaction of poly(ADP- ribose) polymerase as derived from crystal structures
and mutagenesis. J. Mol. Biol. 278:57-65
Ruf A, de Murcia G, Schulz GE. 1998. Inhibitor and NAD+ binding to poly(ADP-ribose)
polymerase as derived from crystal structures and homology modeling. Biochemistry
37:3893-900
Ruf A, Rolli V, de Murcia G, Schulz GE. 1998. The mechanism of the elongation and
branching reaction of poly(ADP- ribose) polymerase as derived from crystal structures
and mutagenesis. J. Mol. Biol. 278:57-65
Satoh MS, Lindahl T. 1992. Role of poly(ADP-ribose) formation in DNA repair. Nature
356:356-8
Schlicker A, Peschke P, Burkle A, Hahn EW, Kim JH. 1999. 4-Amino-1,8naphthalimide: a novel inhibitor of poly(ADP-ribose) polymerase and radiation
sensitizer. Int. J. Radiat. Biol. 75:91-100
Schraufstatter IU, Hyslop PA, Hinshaw DB, Spragg RG, Sklar LA, Cochrane CG. 1986.
Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADPribose) polymerase. Proc. Natl. Acad. Sci. U. S. A 83:4908-12
Schraufstatter IU, Hinshaw DB, Hyslop PA, Spragg RG, Cochrane CG. 1986. Oxidant
injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase
and lead to depletion of nicotinamide adenine dinucleotide. J. Clin. Invest 77:1312-20
139.
Scrofani SD, Wright PE, Dyson HJ. 1998. 1H, 13C and 15N NMR backbone assignments
of 25.5 kDa metallo-beta- lactamase from Bacteroides fragilis. J. Biomol. NMR 12:201-2
Scrofani SD, Wright PE, Dyson HJ. 1998. The identification of metal-binding ligand
residues in metalloproteins using nuclear magnetic resonance spectroscopy. Protein Sci.
7:2476-9
Scrofani SD, Wright PE, Dyson HJ. 1998. 1H, 13C and 15N NMR backbone assignments
of 25.5 kDa metallo-beta- lactamase from Bacteroides fragilis. J. Biomol. NMR 12:201-2
Secrist JA, III, Barrio JR, Leonard NJ. 1972. A fluorescent modification of adenosine
triphosphate with activity in enzyme systems: 1,N 6 -ethenoadenosine triphosphate.
Science 175:646-7
Sekine A, Fujiwara M, Narumiya S. 1989. Asparagine residue in the rho gene product is
the modification site for botulinum ADP-ribosyltransferase. J. Biol. Chem. 264:8602-5
Simonin F, Hofferer L, Panzeter PL, Muller S, de Murcia G, Althaus FR. 1993. The
carboxyl-terminal domain of human poly(ADP-ribose) polymerase. Overproduction in
Escherichia coli, large scale purification, and characterization. J. Biol. Chem. 268:1345461
Simonin F, Poch O, Delarue M, de Murcia G. 1993. Identification of potential active-site
residues in the human poly(ADP- ribose) polymerase. J. Biol. Chem. 268:8529-35
Sims JL, Berger SJ, Berger NA. 1981. Effects of nicotinamide on NAD and poly(ADPribose) metabolism in DNA- damaged human lymphocytes. J. Supramol. Struct. Cell
Biochem. 16:281-8
140.
Siuzdak G. 1994. The emergence of mass spectrometry in biochemical research. Proc.
Natl. Acad. Sci. U. S. A 91:11290-7
Smith S, Giriat I, Schmitt A, de Lange T. 1998. Tankyrase, a poly(ADP-ribose)
polymerase at human telomeres. Science 282:1484-7
Steer BA, Merrill AR. 1994. The colicin E1 insertion-competent state: detection of
structural changes using fluorescence resonance energy transfer. Biochemistry 33:110815
Tweten RK, Barbieri JT, Collier RJ. 1985. Diphtheria toxin. Effect of substituting
aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. J. Biol. Chem.
260:10392-4
Vandekerckhove J, Schering B, Barmann M, Aktories K. 1987. Clostridium perfringens
iota toxin ADP-ribosylates skeletal muscle actin in Arg-177. FEBS Lett. 225:48-52
Wick MJ, Hamood AN, Iglewski BH. 1990. Analysis of the structure-function
relationship of Pseudomonas aeruginosa exotoxin A. Mol. Microbiol. 4:527-35
Wick MJ, Frank DW, Storey DG, Iglewski BH. 1990. Identification of regB, a gene
required for optimal exotoxin A yields in Pseudomonas aeruginosa. Mol. Microbiol.
4:489-97
Wick MJ, Frank DW, Storey DG, Iglewski BH. 1990. Structure, function, and regulation
of Pseudomonas aeruginosa exotoxin A. Annu. Rev. Microbiol. 44:335-63
141.
Appendix A
Composition of growth media and transformation buffer:
(a)
Nutrient agar
one litre of 2X YT nutrient agar contained the following:
bacto agar
15 g
bacto tryptone
16 g
yeast extract
10 g
sodium chloride
5g
The solution was adjusted to pH 7.0 with NaOH and then the preparation was
autoclaved for 25 min at 121º C. Once the solution was cooled to 50º C, the liquid agar
was supplemented with ampicillin (100 g/mL) and was aseptically distributed to sterile
petri plates (approximately 15 mL) and cooled to room temperature.
(b)
2X YT medium
The composition of the 2X YT medium was similar to that described above, except for
the omission of the bacteriological agar.
(c)
Super LB Broth
One litre of super LB broth contained the following:
yeast extract
5g
bactotryptone
3g
142.
sodium chloride
10 g
magnesium sulfate
0.2 g
The solution was adjusted to pH 7.0 with NaOH and then the preparation was autoclaved
for 25 minutes at 121º C.
(d)
SOB media
One liter of SOB media contained the following:
bacto-tryptone
yeast extract
sodium chloride
20 g
5g
0.5 g
An aliquot (10 mL) of a KCl solution (250 mL) was added to the above ingredients in
approximately 900 mL ddH2O and the pH was adjusted to 7.0. The volume was adjusted
to one liter and the solution was autoclaved for 25 minutes at 121º C.
(e)
buffer 1
RbCl
100 mM
MnCl2
50 mM
potassium acetate
30 mM
CaCl2
10 mM
glycerol
15% (w/v)
The solution was adjusted to pH 5.8 with acetic acid and sterilized by filtration.
(f)
buffer 2
RbCl
10 mM
MOPS
10 mM
CaCl2
10 mM
143.
glycerol
15% (w/v)
The solution was adjusted to pH 6.8 with NaOH and sterilized by filtration.
(g)
Lysis buffer
glucose
50 mM
Tris-HCl
25 mM
EDTA
10 mM
The solution was adjusted to pH 8.0 using NaOH.
(h)
(i)
alkaline buffer
NaOH
0.2 M
SDS
1%
buffer 3
ammonium acetate
(j)
7.5 M
Media A
The composition of media A was similar to that described for 2X YT, except for addition
of the following to the solution.
(k)
choroamphenicol
30 g/mL
ampicilin
100 g/mL
salt buffer
NaCl
300 mM
Tris.HCl
100 mM
EDTA
1 mM
The solution was adjusted to pH 8.0 using NaOH.
144.
Appendix B
Compositions of buffers used in the Qiagen plasmid purification procedure. Data from
QIAGEN® plasmid handbook, Feb. 1995, Qiagen Inc. Chatsworth CA.
Composition of buffers
Buffer P1:
(Resuspension buffer)
50 mM Tris-HCl, pH 8.0; 10 mM EDTA;
100 g/mL RNase A
Storage
4 ºC
Buffer P2:
(Lysis buffer)
200 mM NaOH, 1% SDS
room temperature
Buffer P3:
(Neutralization buffer)
3.0 M potassium acetate, pH 5.5
room temperature
or 4 ºC
Buffer QBT
(Equilibration buffer)
750 mM NaCl; 50 mM MOPS, pH 7.0;
15% ethanol; 0.15% Triton X-100
room temperature
Buffer QC
(Wash buffer)
1.0 M NaCl; 50 mM MOPS; pH 7.0;
15% ethanol
room temperature
Buffer QF
(Elution buffer)
1.25 M NaCl; 50 mM Tris-HCl; pH 8.5;
15% ethanol
room temperature
TE
10 mM Tris-HCl; pH 8.1; 1 mM EDTA
room temperature
145.
Appendix C
Electrospray mass spectrometry results of PE24 muteins indicating the
presence of cysteine residue.
Figure E1. ESMS data of the PE24 variant S449C.
The peak corresponding to the PE24 variant appears at
24546 kDa. The peak at 24853 kDa represents the unreactive
species contaminating this preparation of the protein.
146.
147.
148.
149.
150.
151.
152.
153.
154.
Appendix D
The first task in these types of binding studies is to convert the spectroscopic
titration curve (a change in the monitored signal as a function of the titrant concentration)
into a thermodynamically rigorous, model independent binding isotherm, which can then
be analyzed, using an appropriate binding model to extract binding parameters. The
fluorescence emission of PE24-AEDANS is quenched upon binding of the fluorescein
adduct of EF-2 due to energy transfer, hence this signal was used to monitor the
interaction. Therefore, in this case, the observed fractional protein fluorescence
quenching, F/F0 was plotted as a function of EF-2AF concentration.
A detailed study on PE24 interaction with EF-2 was initiated. Typical binding
isotherm is shown in Figure 31. Under experimental conditions, the concentration of
complexes formed is significant as compared with total protein concentration. For a
simple reaction,
Ligand + Receptor  LigandReceptor
where PE24 and EF2 are receptor and ligand, respectively, and the protein-protein
complex is LigandReceptor. At equilibrium, protein complex forms at the same
rate as it dissociates.
[PE24] [EF-2] Kon = [PE24-EF2] koff
The rearrangement of above equation defines the equilibrium dissociation constant
(1)
[EF-2] [PE24]/[EF-2PE24] = Koff/kon = Kd
155.
The law of mass action predicts the fractional receptor (PE24) occupancy at equilibrium
as a function of ligand (EF-2) concentration. Fractional occupancy is the fraction of all
receptors that are bound to ligand.
(2)
Fractional occupancy = [EF-2PE24]/ [PE24]total
Fractional occupancy = [EF-2PE24]/ [PE24] + [EF-2PE24]
Combination of equations 1 and 2 create a useful equation 3 that describes the binding
interaction between PE24 and EF-2 and allows determination of Kd.
(3)
Fractional occupancy = [ EF-2]/[EF-2] + Kd
Equilibrium specific binding at a particular EF-2 concentration equals fractional
occupancy times the total PE24 concentration (receptor number B max):
(4) Fractional occupancy. B max = specific binding = Bmax . [EF-2]/Kd + [EF-2]
The above equation describes the binding isotherm for PE24 and EF-2 interactions.
Thus, the value of Kd can be obtained by fitting the experimental data to the equation by
using nonlinear regression analysis.
It is important to consider the assumptions made in the above binding model. The
first assumption is that binding follows the law of mass action and has equilibrated.
Second, it is assumed that there is only one population of PE24 and that only a small
fraction of EF-2 binds so that the free concentration is essentially identical to the
concentration added. Finally, it is assumed that no co-operativity exists in binding. In
other words, the Kd is constant during the experiment.