Identification and Characterization of a Second Catalytic Glutamate within
the Active Site of Pseudomonas aeruginosa Exotoxin A that is
Responsible for Transferase Activity‡
Xu Wang, Xiaobo Liu, Gerry A. Prentice, Rene Jorgensen and A. Rod Merrill†
From the Department of Molecular and Cellular Biology, University of Guelph, Guelph,
Ontario N1G 2W1, Canada
Running title: catalytic loop in P. aeruginosa exotoxin A
†
To whom correspondence should be addressed. Tel.: (519) 824-4120 ext. 53806; Fax: (519)
837-1802, Email:rmerrill@uoguelph.ca
‡
Supported by the Canadian Institutes of Health Research, A.R.M.
Draft: Dec 72, 2005
Formatted for J. Biol. Chem.
1
SUMMARY
The bacteria causing diphtheria, whooping cough, cholera and other diseases secrete
mono-ADP-ribosylating toxins that modify intracellular proteins in the target, host eukaryotic
cell. Recently, we solved four high-resolution crystal structures of a catalytically active
complex between the enzyme domain of Pseudomonas aeruginosa exotoxin A (ETA) and its
protein substrate, translation elongation factor 2 (eEF2), which has led to a breakthrough in
the understanding of the reaction mechanism of this family of deadly toxins. The target
residue in eEF2, diphthamide (a modified histidine), spans across a cleft in the proteinprotein complex and faces the two phosphates and a ribose of the non-hydrolysable NAD+
analogue, TAD. This suggests that the diphthamide is involved in triggering NAD+ cleavage
and interacting with the proposed oxacarbenium intermediate during the nucleophilic
substitution reaction, explaining the requirement of diphthamide for ADP ribosylation. The
structures, however, do not reveal the transition state for the transferase reaction within the
complex. We have new mutagenesis and kinetic data that implicate new catalytic residues
located within an active site loop (Loop 3) within ETA, but not within the corresponding loop
of diphtheria toxin. Loop 3 does not interact directly with either the diphthamide or NAD+ in
the X-ray structures. It is proposed that this loop functions as a lid for the active site to
restrict solvent access to the reaction center during catalysis.
The lid is in an open
conformation in the pre-transition state structures, and during transition-state formation, it
closes in on the active site partly as a cover for solvents and partly to help stabilize the
oxacarbenium ion. It is proposed that the hinge action of the lid activates further steps in the
reaction mechanism involving the migration and orientation of the diphthamide nucleophile
in a position adjacent to the oxacarbenium reactive cation species.
INTRODUCTION
Bacterial virulence factors are moieties that are produced by bacterial pathogens
essential for causing disease in a host. An important class of virulence factors is the bacterial
exotoxins that are secreted by viable pathogenic cells. Toxins play an important role in the
various strategies developed by pathogenic bacteria to cause disease, since these proteins are
responsible for the majority of symptoms and lesions during infection (1) These bacterial
proteins are amongst the most potent toxins known to man. The majority are A-B or binary
toxins that bind to the target membrane with a receptor-binding domain (B subunit) and
deliver a second moiety (A subunit) into the cytoplasm. One large group of A-B toxins is
known as the mono-ADP-ribosyltransferase family (ART). The ARTs are enzymes that act
2
to kill target eukaryotic cells by covalent modification of specific proteins within the host
organism. The general reaction scheme for the ART family of enzymes is shown in Figure 1.
These enzymes bind NAD+, facilitate the scission of the glycosidic bond (C-N) between
nicotinamide and the N-ribose of NAD+, and transfer the ADP-ribose group to a specific
target protein within the host cell. The reaction is believed to follow an SN1 nucleophilic
substitution mechanism (2-4), despite the observed inversion of configuration of the C-N
bond of the ribosyl-substrate product (5;6). In addition, this family of enzymes also possesses
NAD+ase or glycohydrolysis activity, but the physiological relevance of this activity is not
known (7;8).
The best characterized proteins of this toxin family are cholera toxin (CT) produced by
Vibrio cholerae, heat-labile enterotoxin (LT), from E. coli, pertussis toxin of Bordetella
pertussis (PT), diphtheria toxin (DT) produced by Corynebacterium diphtheriae, and
exotoxin A (ETA) of Pseudomonas aeruginosa. A second group of related proteins includes
Clostridium botulinum C2 toxin (9) C. perfringes iota toxin (10), and C. difficile toxin (11)
that use actin as the acceptor molecule and interfere with its polymerization. Remarkably, all
the known members of this family catalyze the same enzymatic reaction (Figure 1) despite
the observation that there is no significant or extended sequence homology among the family
members. However, even though the sequence similarity is limited there is still a common
core fold of ~100 residues that contains the NAD+ binding site within the ART enzymes (1216). The common fold represents a structural motif for binding this nucleotide cofactor that is
unique to this family of toxins and is unlike the Rossmann fold that is characteristic of the
dehydrogenases (14). It consists of two antiparallel -sheets and two -helices with the active
site cleft formed at the interface of the two -sheets. In addition, elegant biochemical
experiments involving photoaffinity labeling and site-directed mutagenesis conducted by
Collier and others have demonstrated that these toxins possess a catalytic glutamate that is
critical for enzymatic activity (17-22). The structural identity of the active site has later been
confirmed by the X-ray structures of several enzymes (13;23-27).
The human pathogen, P. aeruginosa produces ETA, which is a 66-kDa extracellular
protein that is internalized into the eukaryotic cell during bacterial infection (Yates et al.,
TiBS in press). Based on the crystal structure of full-length, intact ETA (28;29), it consists of
three distinct functional domains. Domain I is involved in receptor binding, domain II aids in
translocation across the membrane into the host cell cytoplasm after receptor-mediated
endocytosis (30) and domain III comprises the catalytic domain containing an extended cleft
3
that serves as the enzyme’s active site. Similar to ETA, DT from C. diphtheriae is secreted as
a single 58-kDa three-domain polypeptide and is also cleaved into two fragments before or
during the binding of the epidermal growth factor (EGF)-like growth factor precursor (HB–
EGF) that function as the DT receptor (31). Like ETA, DT is taken into the host cell by
receptor-mediated endocytosis, but it reaches the cytoplasm earlier than ETA by penetrating
through the membrane in the early endosome (Yates et al., TiBS in press).
Upon gaining access to the host cytoplasm, both ETA and DT catalyze the ADPribosylation of eukaryotic elongation factor-2 (eEF2). eEF2 is a member of the GTPase
superfamily, and is a single polypeptide chain with a molecular mass of ~95 kDa (32). It is a
translation factor responsible for the translocation of tRNA and mRNA on the ribosome
during protein synthesis. Interestingly, eEF2 contains a post-translationally modified histidine
residue,
2-[3-caroxyamido-3-(trimethylammonio)-propyl]histidine,
called
diphthamide
(33;34) which in yeast is located at position 699. The diphthamide is only found in eEF2 and
is completely conserved throughout all eukaryotic and archaebacterial evolution. During
toxin attack, eEF2 is inactivated through the covalent attachment of the ADP-ribosyl moiety
to the N3 atom of the imidazole ring in the diphthamide residue (33;35;36), resulting in
cessation of protein synthesis and eventually cell death (37-39). Importantly, the common
fold region for ETA and DT is structurally indistinguishable despite the low sequence
homology shared between these two toxins. In this motif, NAD+ forms a slightly twisted
horseshoe-shaped structure that has each end of the molecule projecting into the active site
cleft of the C-domain. It forms several H-bonds and hydrophobic contacts with the protein
while the phosphate groups at the base of the horseshoe protrude out of the cleft and are
exposed to solvent (12;13). This kind of strained NAD+ conformation and the solvent
exposure of the NAD+ phosphates is also observed in structures of NAD+ in complex with
VIP2 (25), Iota toxin (26) and the C3 exoenzyme (40). Hence, both the fold and the
presentation of NAD+ to the protein being modified is conserved among these quite diverse
toxins within the family, and therefore these features are likely to be universal to all ADP
ribosylating toxins.
The docking site for the NAD+ substrate within the catalytic domain of ETA has been
well characterized (4;12-14;41). The known catalytic residues within ETA include Glu-553,
His-440, Tyr-481 and Tyr-470. Glu-553 forms a hydrogen bond with the 2’ hydroxyl oxygen
of the N-ribose of NAD+ and maintains the dinucleotide substrate in the proper orientation to
allow exposure of the scissile N-glycosidic bond, and the negatively charged carboxylate of
Glu-553 may stabilize a positively charged reaction intermediate (42). The critical catalytic
4
residue, Glu-553 of ETA and Glu-148 of DT, could stabilize or orient the oxacarbenium
intermediate after dissociation of nicotinamide by forming a hydrogen bond with the 2’OH of
the nicotinamide (N)-ribose, possibly aided by the phenol group of Tyr-481, Tyr-65 of DT
(43-45). Furthermore, it is likely that the strained conformation of the bound NAD+ substrate
in the ART toxins may facilitate cleavage of the glycosidic bond (46). Important new X-ray
structures of the Michaelis complex of eEF2 and ETA indicate that the interaction between
the diphthamide and the NAD+ Ν-phosphate may help to trigger the transferase reaction (46).
It was noted in those structures that the distance between the N-ribose C1 electrophile and the
nucleophilic diphthamide N3 atom observed in the Michaelis complex was too large to be the
transition state (~11 Å). One explanation for this gap could be that the oxarbenium ion
migrates from the NAD+ pocket in ETA towards the diphthamide N3 atom, but that would
obviously require that the highly reactive oxacarbenium ion was protected from reacting with
water and/or nucleophilic residues during transfer across the intermolecular cleft–which is
readily accessible to solvents. Another explanation could be that the gap is reduced by a
substantial rotation of eEF2 relative to ETA in the Michaelis complex, but major
conformational movements seem to be prevented in the enzymatically active crystalline state
by the tight crystal packing around ETA and eEF2 domain IV. A third possibility, and
perhaps the most likely, is that the loop region containing the diphthamide may undergo a
transient conformational change during the reaction, thereby bringing the diphthamide N3
atom closer to the oxacarbenium ion. Notably, the X-ray structure of the product for the
reaction catalyzed by ETA (ADPR-eEF2) confirmed that the nucleophilic substitution
reaction results in an inversion of configuration for the N-ribose C1 bond with the
diphthamide N3 (6).
Although the catalytic residues within ETA and DT are well characterized, the
mechanism of the ADP-ribosylation reaction still remains elusive. In the crystal structure of
the catalytic domain of ETA, a loop region (residues 546-551) near Glu-553 was identified
(12;13) and proposed to act as a clamp to hold the tip of domain IV within eEF2 near the
active site, or perhaps to stabilize the transition state structure (47). More recently, the
involvement of this loop in the catalytic mechanism and in the interaction with the eEF2
substrate was further supported from the crystal structures of the toxin in complex with eEF2
(46). Furthermore, in the cholera toxin group within the ART family, a second catalytic
glutamate/glutamine has been proposed to be involved in the coordination of the nucleophile
(48). In the present study, we survey the function of this active-site loop by alanine-scanning
5
mutagenesis (ASM) of each residue within the ETA sequence. This approach is followed by
multiple residue replacement at two sites within the loop (Glu-546 and Arg-551). In addition,
the corresponding loop in DT (residue 140-146) was also scanned by ASM in an effort to
identify the conserved/essential residues within the region. Both ART and GH enzyme
activities along with NAD+ substrate binding ability of the toxin mutants were measured and
the activities were correlated with the new, high-resolution structure of the complex between
ETA and eEF2 (46).
EXPERIMENTAL PROCEDURES
Overexpression and Purification of ETA and DT—The catalytic 24 kDa fragment of ETA
with a C-terminal 6-His tag was overexpressed and purified as described earlier (8) with only
a slight modification. The catalytic 28 kDa fragment of DT with a C-terminal 6-His tag was
overexpressed and purified as described in (49) with some modification. After harvesting the
cells, the pellet was resuspended into 40 mL of lysate buffer (50 mM Tris, 100 mM KCl,
0.1% Tween-20, 1 mM DTT, 0.5 mM PMSF, pH 8.0) and the cells were lysed in a French
Press (two passes). The lysate solution was then centrifuged at 20,000 x g for 15 min and the
supernatant was collected and filtered through a 0.45 m syringe filter before loading to a 2
mL chelating Sepharose Fast Flow column (Amersham Biosciences AB, Sweden).
Purification of eEF2—Yeast cake (S. cereviseae) was lysed by passing the suspended cells
several times through a High Pressure Homogenizer (Avestin, Inc., Ottawa, ON) at 25,000
p.s.i. and the eEF2 was purified as described (50).
Site-directed Mutagenesis—All the mutants were prepared by the standard QuikChangeTM
mutagenesis protocol (Stratagene, La Jolla, CA).
The DNA template was the plasmid
containing the gene encoding the catalytic domains for wild-type ETA (or wild-type DT) to
prepare the single-site mutants using several sets of primers. The DNA template for double
mutants was the plasmid containing the gene for the single mutant. The desired mutations
were confirmed by DNA cycle sequencing with an Applied Biosystems 3730 DNA Analyzer
(Applied Biosystems, Foster City, CA; CBS DNA Facility, University of Guelph).
Circular Dichroism Spectroscopy–Circular dichroism (CD) analyses were performed with a
Jasco J-715 spectropolarimeter (Jasco, Easton, MD) with samples in a 0.2 mm pathlength,
flat quartz cell. The proteins were scanned from 250 to 180 nm in 20 mM Tris, 50 mM NaF,
at pH 7.8 and an average of four scans was used to generate CD spectra.
Fluorescence Measurements—Fluorescence measurements for ADPRT and GH activities
were obtained using a Cary Eclipse fluorimeter (Varian, Mississauga, ON) equipped with a
6
Peltier-thermostatted multi-cell holder set at 25 °C. Fluorescence emission spectra of toxin
mutants (Trp fluorescence) were collected with a PTI Alphascan-2 T-format fluorescence
spectrometer (PTI Inc, Birmingham, NJ) complete with spectral correction features and the
sample temperature was controlled with a recirculating water bath (25 °C).
Fluorescence-based ADPRT Assay—The NAD+-dependent ADPRT activity of the various
mutant proteins for both ETA and DT was tested as reported previously (47;51). The
excitation and emission monochromators were set to 305 and 405 nm, respectively with 5 nm
bandpasses. The ADPRT reaction contained 20 mM Tris-HCl, pH 7.9, 500 M -NAD+
(Sigma) and eEF2 (at saturating levels) and the reaction progress was monitored in a
disposable ultra-microcuvette (BrandTech Scientific, Essex, CT; 1-cm path length).
-AMP Standard Curve and Assay Calibration—A stock solution of -AMP (Sigma; prepared
in distilled water) was used to prepare a series of standards in 20 mM Tris-HCl, pH 7.9. The
fluorescence of the -AMP standards (0–10 M) was recorded to generate a standard curve
having a slope with units of fluorescence intensity per micromolar -AMP. This slope from
the standard curve was used to convert the slopes obtained for the ADPRT and NAD
glycohydrolase assay measurements to catalytic rates with units of micromolar -NAD+/s.
Quenching of Intrinsic Protein Fluorescence—The NAD+-dependent quenching of the
intrinsic tryptophan fluorescence in the toxins was used to determine the binding constants
(KD) for NAD+ as described (4).
NAD Glycohydrolase Assay—The hydrolysis activity of the various mutants was tested as
detailed previously (8). Briefly, buffer consisting of 20 mM Tris-HCl, 50 mM NaCl, pH 7.9,
-NAD (500 M), and the toxin ETA (final concentration 10 M) were combined together in
a disposal ultra-microcuvette containing 70 L solution (Brand GMBH, Wertheim,
Germany). The fluorescence increase was monitored during the production of -ADP-ribose
at 25C for 4 h and the slope was calculated from the linear fit to the kinetic data.
Sequence and structural alignment of ADPRT family members—The eight ADPRT structures
were initially aligned by using the known catalytic glutamate, arginine or histidine residues,
and the structurally similar elements were identified in their sequences. The same catalytic
residues were marked on the remaining sequences for which this information exists, followed
by alignment-based identification of many similar sequences to identify potential patternfitted
regions
(using
PSI-BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/),
(http://www.ch.embnet.org/software/LALIGN_form.html),
DIALIGN
LALIGN
(http://bibiserv.
techfak.uni-bielefeld.de/dialign/), and ClustalW (http://www.ebi.ac.uk/clustalw/), as well as
7
visual identification. One critical assumption was that a conserved catalytic glutamate is
present in all ADPRT protein sequences. Secondary structure predictions using GOR IV
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html), JPred (http://www.
compbio.dundee.ac.uk/~www-jpred/), PsiPred (http://bioinf.cs.ucl.ac.uk/psipred/) and HNN
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html)
algorithms
were
performed on sequences containing two or more possible central patterns, such as the
dinitrogen reductase (DRAT), to identify the best candidates. Sequences for the universal
standard proteins (-galactosidase, ovalbumin, and alkaline phosphatase) were also examined
as controls, and none fit the pattern using the same search criteria. Rendering of the 3-D
models of the protein structures and structural alignment of the loop regions within the
ADPRT enzymes was accomplished with DSVIEWER Pro 5.0 (Accelrys, San Diego, CA).
RESULTS
Briefly, all the ADPRT enzymes can be divided into two groups: the DT group,
composed mainly of DT, ETA, and poly (ADP-ribose) polymerase (PARP) (included here
despite the distinction that these enzymes are poly-ADPRTs) and the CT group comprising
CT, LT, PT, Clostridium C3 exoenzyme (C3), mosquitocidal toxin (MTX), and others
(Figure 2). The amino acid residue on the target protein functions as the nucleophile for the
reaction and includes diphthamide (modified histidine) and glutamate for the DT group and
cysteine, asparagine, and arginine for the CT group (Figure 2).
The consensus motif for the ADPRT family shows that the active site is formed in these
enzymes by three short, structurally conserved regions (Figure 2). Region 1 is characterized
by a catalytic His or Arg residue (DT and CT groups, respectively) and is reasonably
conserved between the two groups. In the CT group, region 1 extends in the N-terminal
direction to 2 conserved amino acids, where the first is hydrophobic (Leu or Val) and the
second is aromatic (Tyr or Phe). Region 2 makes the scaffold of the active site and forms a
beta strand followed by a slightly tilted -helix that varies in length in the toxins (12 residues
in DT, ETA, and LT, to 21 residues for PT). A conserved Tyr residue in the DT group helps
to form the nicotinamide binding pocket for the NAD+ substrate and the same function is
performed by an aromatic residue within the CT group (Figure 2). Region 3 contains the
catalytic Glu (Glu-148 of DT, Glu-553 of ETA, Glu-112 of CT and LT, and Glu-129 of PT).
These amino acids are located in virtually identical positions within the active site cleft,
facing opposite to the Arg/His residue on an antiparallel -strand close to the external face of
the catalytic domain. In the CT group, this region can be extended to include a Glu/Gln
8
residue in position -2, which gives the pattern Glu/Gln–X-Glucat (25;40;48). Importantly, the
essential role in catalysis of the conserved residues within regions 1 and 3 and the structure of
the active site cavity in region 2 has been verified by site-directed mutagenesis (19;20;52-54).
The core X-ray structures of a few representative members of both groups of the monoADPRT family are shown in Figure 3, including the chicken PARP structure (for
comparison). It is clear that the catalytic core region of this enzyme family is structurally
conserved despite the lack of sequence similarity (Figs. 2 and 3). The catalytic Glu (red in
Figure 3, region 3) is invariably located on a beta strand (orange) that pairs up with a second
beta strand (blue in Figure 3, region 2), the latter strand is known to contain the essential Tyr
(DT group) or aromatic (CT group) stacking to the nicotinamide. Thus, this beta strand and
the accompanying -helix in region 2 serve to “frame” the active site cavity into which the
nicotinamide ring of NAD+ enters and is anchored during the reaction. The - structure of
region 2 has little or no amino acid sequence homology among family members. However, it
can be identified by an F/Y-X-S-X-S/T/Q motif in the enzyme core of the CT group, and by
G-F/L/I-Y-(X)10-Y in the DT group (48). The beta strand within region 1 (shown in grey) is
oriented nearly perpendicular to the aforementioned beta pair in the catalytic core and houses
the conserved aromatic residue (green) and catalytic His (DT group) or Arg (CT group)
residues (pink in Figure 3).
Alanine scanning mutagenesis of Loop 3—Earlier, it was proposed that the loop region
connecting beta strand 4 with strand 5 in the ADPRT catalytic core is responsible for the
target substrate (protein) specificity (Koch-Nolte, Bazan, 2001) and in our quest to elucidate
the details of the catalytic mechanism for the mono-ADPRT enzyme family, we chose to test
this hypothesis by performing Ala scanning mutagenesis of this loop region (Loop 3) within
ETA (Figure 4A). The kinetic data for the Ala scan of Glu-546–Glu-553 are shown in Table
1. The relative ADPRT and GH enzymatic activities for the WT and mutant ETA enzymes
are shown along with the binding affinity of these enzymes to the NAD+ substrate. In order
to ascertain whether the effect of residue substitution was due to change in the residue
chemistry or whether the effect was due to a loss in the folded integrity of the ETA enzyme,
the Trp fluorescence  emission maxima (em,max) were measured for the WT and mutant
enzymes (Table 1). The WT ETA protein possesses a Trp em,max near 331 nm and none of
the mutant enzymes exhibited shifts in their Trp em,max values that were greater than 3 nm
(330-333 nm) indicating that the point mutations did not significantly alter the folded
integrity of the mutant enzymes. Circular dichroism spectra were collected to determine the
9
solution secondary structure for a select group of the less active enzymes, including E546A,
E553A, and E546/R551A and the secondary structure was also found to be preserved and
was very similar to the WT spectrum (data not shown). Previously, we demonstrated that
there is a strong correlation between the Trp em,max and the secondary structure as
determined by circular dichroism spectroscopy (4;41;55).
It is readily apparent from the
ADPRT kinetic data that two residues within ETA are critical for ADPRT activity (Glu-546
and Glu-553), which showed only 0.12 and 0.l5% of WT activity, respectively, when
replaced with Ala residues. The latter Glu residue has been well characterized and is known
as the most critical catalytic residue within this enzyme family (43;44;56); however, the
former residue has not previously been implicated in the catalytic mechanism for the DT
group of ribosyltransferases. Remarkably, the effect of Ala substitution at residue 546 did
not impair NAD+ substrate binding or GH activity nearly as much as the E553A replacement,
indicating that the Glu-546 and Glu-553 residues are participating in different aspects of the
catalytic mechanism. Glu-553 participates in the catalytic process by forming an H-bond with
the 2’-OH of the nicotinamide ribose of NAD+, which destabilizes the glycosidic C-N bond
while stabilizing the oxacarbonium transition state and this facilitates the nucleophilic attack
on the N-ribose anomeric carbon (C1) by N3 of diphthamide (12;13). Importantly, Glu-546
is clearly acting in a different component of the catalytic mechanism and is likely involved in
facilitating the transferase step in the reaction, a function that has been sought after as the
Holy Grail for this reaction mechanism, but has so far proved elusive (54).
There was also a minor effect on the ADPRT activity caused by the R551A mutation
(38% of WT ADPRT activity), with Gly-550 and Glu-547 also showing a slight sensitivity to
Ala substitution (56 and 77% of WT ADPRT activity, respectively; Table 1). Multiple
substitutions at the Glu-546 site were then conducted in order to try to understand the
underlying cause of this significant loss in ADPRT activity with only a modest effect on both
NAD+ substrate binding and GH activity. Table 1 shows that only the replacement of Glu546 with Gln could partially restore the lost ADPRT activity (1.1% of WT). Surprisingly, the
replacement of Glu with Asp at residue 546 resulted in a less active mutant enzyme than the
Gln replacement, indicating the importance of the position of the carboxylic group of Glu546 in the reaction mechanism.
Multiple substitutions at Arg-551 showed that the ADPRT activity of ETA was
sensitive to the nature of the substitution (Ala > Lys > Gln > Glu > His), however, these
effects were likely due to electrostatic or packing effects and not related to the direct
10
involvement of Arg-551 in the reaction mechanism, since the R551A mutation still showed
considerable ADPRT activity (38%, Table 1). A close inspection of the X-ray structure of
the catalytic complex (ETA:TAD:eEF2)(46), suggests that Arg-551 plays an ancillary role
in the catalytic mechanism, possibly to H-bond with Glu-546 and may help to tether this
residue in the proper orientation for catalysis (Figure 4A), or alternatively to work in concert
with Glu-546 to position the catalytically crucial Glu-553 residue.
Importantly, the
replacement of Arg-551 with a negatively charged Glu residue drastically reduced the
ADPRT activity (1.1 %, Table 1), indicating the possible involvement of electrostatic
interactions at the 551 site for the conformation of Loop 3 and its putative role in the
positioning of Glu residues, 546 and 553. Furthermore, the replacement of Arg-551 with the
neutral imidazolium ring of His caused nearly a total loss of enzyme activity, which we
attribute to a perturbation of the orientational integrity of Loop 3 within the active site core
that may lead to a disruption of the precise positioning of Glu-546 and possibly also Glu-553
in the Michaelis complex (Table 1, Figure 4A).
Evidence for the concerted involvement of both Glu-546 and Arg-551 in the catalytic
mechanism of ETA is found in the effect of the double Ala mutant, E546A/R551A, which
showed almost complete loss of ADPRT activity, yet showed near WT activity for NAD +
binding and GH activity (Table 1). The more conservative double replacement mutant,
E546D/R551K, did little to restore the lost ADPRT activity (0.09%) and the residue reversal
mutant, E546R/R551E, was also nearly devoid of ADPRT function (0.0001%) (Table 1).
The importance of Glu-546 within Loop 3 in the catalytic mechanism of ETA
suggested the possibility that this may be a conserved element within the large and diverse
mono-ADPRT family of the enzymes. A search for a comparable catalytic element in some
mono-ADPRT family members having high-resolution X-ray structures (DT, PARP, C3
exoenzyme, PT, CT, vegetative insecticidal protein 2, iota toxin, and E. coli heat-labile toxin)
showed that such a motif could be identified (Table 2). The loop length varied from 6
residues (ETA) to 37 residues (PARP) and a pair of H-bonding partners was identified within
the loop motif. However, there is no conservation of the Glu-546 residue within these
enzymes, indicating that either this transferase function is unique to ETA or that the
transferase function was provided by another residue within the motif or another element
within the catalytic core of the related enzymes. The closest relative of ETA is DT, which
also recognizes and modifies eEF2. Consequently, an in-depth comparative analysis of both
ETA and DT was conducted.
11
A sequence alignment of Loop 3 from ETA with DT revealed that there is
considerable similarity and certainly some identity between the two corresponding loop
elements (Figure 4C). The loop within DT is one residue longer (Table 2) but does not show
any alignment with Glu-546 from ETA. However, DT possesses a Glu residue at position
142, which aligns with Glu-548 of ETA, suggesting the possibility of a positional shift within
the loop for the functional Glu residue within DT. This was further investigated by sitedirected mutagenesis of Glu-142, Phe-140, Ser-144, Ser-145 and Ser-146 in the DT loop
region. Replacement of Glu-142 with either Ala or Asp had little or no effect on the ADPRT
activity of the DT enzyme (Table 3), nor was there any significant effect on the NAD+
binding or GH activities of these mutations, except that the ability of the E142A and E142D
to bind the NAD+ substrate was surprisingly enhanced by 160 and 240%, respectively.
Replacement of Ser-146 with either Ala or Thr had a slightly greater inhibitory effect on the
ADPRT activity of DT than replacement at Glu-142, but the magnitude of the effect was still
small (Table 3). The mutations with the most effect on both ADPRT and GH were the F140P
and S146P mutations ranging from a 50-330 fold reduction in ADPRT activity but only about
2-fold reduction in GH activity.
The Loop 3 motifs (5-loop-6) from the known ETA x-ray structures were aligned to
determine if there were significant changes in the disposition of any of the motif elements or
its constituent residues, Glu-546, Arg-551, and Glu-553 (Fig 5A). The disposition of Loop 3
does change in the various complexes but generally the positions of the three residues do not
vary significantly. Furthermore, alignment of the corresponding Loop 3 elements among 5
ADPRT members, C3, ETA, DT, and PARP was also performed to compare this motif within
the family (Figure 5B). PARP possesses a much longer loop than any of the other members,
which may or may not be attributed to its unique ability to catalyze the polymerization of
ADP-ribose units during its catalytic cycle (Smith, 2001). From the alignment it is clear that
DT and ETA possess the most similar Loop 3 motifs in both size and conformation, while the
C3 exoenzyme shows a markedly different loop disposition.
DISCUSSION
Our previous study implicated a loop region within ETA (previously designated Loop
C, but now designated Loop 4 (46)) and more specifically, a small subregion within this loop
(Gln-483, Asp-484 and Asp-488), that is an essential catalytic element in the ADPRT
reaction mechanism (54). It was previously suggested that Loop 4 is an important catalytic
12
element within ETA since the substrate binding data (NAD+- and eEF2 substrates) and the
steady state kinetic parameters, KM(-NAD+) and KM(eEF2), were unaffected by Loop 4
replacement, yet the kcat was reduced by 20,000 fold. In the structure of the catalytic domain
of ETA, the phenol ring of Tyr-481 stacks with the nicotinamide ring of NAD+ near the site
of cleavage where the ADP-ribosyl group of NAD+ is transferred to eEF2 (12;13). Therefore,
residues involved in this transfer event must be spatially situated near Tyr-481.
An
examination of the position of the subregion (in particular, residues Gln-483, Asp-484, and
Gln-485), correlates with this notion since they are on one face of Loop 4 in close proximity
to Tyr-481 and are located close to the NAD+ substrate. However, the remaining residues
within Loop 4 are situated on the opposite side of the loop and are more distant from the site
of the reaction. Asp-488 is important for activity; however, it is not situated as closely to
Tyr-481 as the other catalytically important residues in question. However, the kinetic data
for the double Asp mutant enzyme showed that Asp-488 acts in concert with Asp-484.
Unfortunately, the distance between these two residues is too great for any direct interaction,
but these residues could be linked through a bridged water molecule (however, it was not
resolved as a heteroatom in the structure (13)) since the X-ray structure shows Asp-488
participating in several hydrogen bonds. Therefore, it was proposed that Asp-488 may play a
structural role within the Loop 4 region by properly aligning those residues, in particular Gln483, Asp-484 and Gln-485, that are, perhaps, involved in the stabilization of the transition
state for the ADPRT reaction, which would involve parts of both the NAD+ and eEF2
substrates as the kinetic data suggest.
The X-ray structures of the ETA-eEF2 complexes (46) indicate that Loop 3 does not
directly contact the eEF2 substrate as previously proposed (57), but rather Loop 4 is the
primary point of contact between the two proteins. However, it is also clear from our 4 Xray structures of the complex (apo-, TAD-, PJ34-, and ADP-ribose-complex) that there are
additional steps in the catalytic mechanism that have yet to be identified (46) and which
could involve direct interactions with Loop 3. Nonetheless, Loop 3 does make an important
connection between two beta strands, one of these strands harbors the essential Glu-553
residue (Figure 4A) and the position of this beta strand and its Glu-553 tenant are conserved
within the catalytic core of the ADPRT enzymes (Figure 3). The remarkable sensitivity of
the ADPRT function of the ETA enzyme to substitution at Glu-546 indicates that Loop 3, and
in particular, Glu-546, also participates in the catalytic mechanism of the enzyme. Glu-546
does not likely participate in the scission of the glycosidic bond between the C1 of the
13
nicotinamide ribose and nicotinamide N1 of NAD+ because replacement of Glu-546 does not
have a large effect on the GH activity or NAD+ substrate binding ability of the enzyme (Table
1), which differs from the effect of replacement of the catalytic, conserved Glu-553 residue
(Table 1) (43;44;56). Substitution of Arg-551 has a notable effect on ADPRT activity,
depending on the nature of the replacement residue. Residue 551 is positioned too far from
the active site to participate directly in the catalytic mechanism, but forms a hydrogen bond to
Glu-546. Therefore, it is possible that Arg-551 is important for stabilization of Loop 3 and for
the correct positioning of Glu-546 in its catalytic role. It is interesting, however, that the
R551E mutation in ETA results in a dramatic reduction in ADPRT activity since the highly
conserved putative toxA from V. cholera (58) actually has a Glu residue at the position
equivalent to Arg-551 (Fig. 2).
Surprisingly, the ASM of Loop 3 of the DT did not reveal any obvious catalytic
residues. The residues that were most sensitive to mutation were F140 and S146, which forms
a main chain hydrogen bond in the DT structure (1DTP) similar to the 546-551 hydrogen
bond in ETA. Replacement of the Phe-140 and Ser-146 residues of DT with a proline residue
most likely invokes a kink or bend within the loop conformation, disrupting the hydrogen
bond between these residues and disrupting the conformation of Loop 3. The disruption of
the hydrogen bond may impact the function of Glu-148, which may account for the decrease
in catalytic activity, however, Loop 3 in DT does not appear to contain a second catalytic
residue as seen in the corresponding loop of ETA. This may also explain why the effect of
residue substitution for Arg-551 in ETA with residues that may disrupt the hydrogen bond
between Arg-551 and Glu-546 also affects the enzyme activity, since the ADPRT activity of
ETA also depends on Glu-546.
One possible function of Glu-546 may be to participate in the transfer component of
the ADPRT reaction by facilitating the movement of the ADP-ribose oxacarbenium ion
towards the nucleophilic diphthamide residue, since this Glu residue is clearly physically
situated enroute in the reaction pathway between the electrophile (oxacarbenium ion) and
nucleophile (diphthamide of eEF2) (Figure 4A) (46). It is clear that the double mutant,
E546A/R551A, provides an enzyme that has lost its ability to transfer ADP-ribose to eEF2
(diphthamide) but can still interact with solvent OH- (Table 1). Thus, Glu-546 may be an integral
part of the essential catalytic “annealing” mechanism whereby the 10–11 Å gap between the
electrophile and nucleophile is reduced during the catalytic cycle. On the other hand, one
might also question whether such a movement of the highly unstable oxacarbenium species
through what seems to a very solvent accessible pathway is possible at all.
14
Another explanation, that is perhaps more likely, could be that Loop 3 serves as a lid
for the active site of the ADPRT enzymes. In this model, Loop 3 is in an open conformation
in the complex structures elucidated so far (46), and during transition-state formation, the
Loop 3 lid would close in on the active site, partly to eliminate solvent access to the reaction
centre and hence to increase the lifetime of the glycofuranosyl oxacarbenium ion species, and
partly to position Glu-546 for H-bond formation with the amide substituent of the nucleophile
(2-position substituent of the diphthamide imidazole ring) in the eEF2 substrate. In this
model, the hinged motion of Loop 3 would facilitate the approach and orientation of the
diphthamide nucleophile (N3 atom) toward the oxacarbenium C1 ribose cation. This notion
of a substrate-recognition function for Glu-546 is not unique, since a similar role has been
proposed for Gln-212 in Clostridium botulinum C3 exoenzyme in the recognition of Asn-41
in its Rho protein substrate (40). Furthermore, a similar mechanism involving a loop
movement during transition-state formation has previously been shown in crystal structures
of a purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor
(59;60). Phosphoribosyltransferases catalyze a similar reaction in the nucleotide synthesis
salvage pathway involving an oxacarbenium ion that also seems to be stabilized by
interactions involving one or two adjacent and highly conserved acidic residues with the
hydroxyl groups of the ribose involved in the reaction (3;59-62). At present, we do not have
an explanation for the details of the proposed annealing step(s) in the ADPRT catalytic
mechanism, but must await further elucidation of reaction complexes by both kinetic analysis
and X-ray crystallographic work, currently in progress in our laboratory
In summary, a working model for the ADPRT reaction mechanism is proposed that
provides a plausible explanation for the recent mutagenic, kinetic and crystallographic data
for the DT group of enzymes. Initially, a ternary complex forms between ETA, NAD+ and
eEF2, which can assemble in random order (2;46). Our new high-resolution structures and
kinetic measurements indicate an initial Michaelis complex that features a relatively large
distance between the electrophile (NAD+ C1) and the nucleophile (diphthamide N3), which
supports the proposed SN1 mechanism for ADP-ribosylation and infers additional steps in the
reaction mechanism. Docking and binding of the NAD+ substrate within the enzyme’s active
site results in a strain exerted upon the substrate glycosidic C-N bond causing it to weaken.
These precursor binding event(s) result in a loss of the C-N bond order, which initiates the
partial leaving of the nicotinamide moiety invoking a structural change within the catalytic
complex that likely features the interaction of the diphthamide moiety within the eEF2
protein substrate (Diph-699 in the yeast protein) with catalytic residues found in loop
15
elements of the enzyme. An important step in this catalytic model may be the interaction of
Glu-546 with the diphthamide ammonium and/or amide substituent, which may then draw
both NAD+ phosphates into range for electrostatic interaction with the diphthamide
ammonium group, helping to explain the essential role that the diphthamide residue plays in
catalysis. In the pre-transition state of the Michaelis complex for ETA, there are only a few
candidate residues that may function as participants in the transferase reaction. Asn-581
within eEF2 is tightly engaged in a hydrogen bond with eEF2 Gln-704 and the amide group
of the diphthamide moiety. Also, the two nitrogen atoms of the diphthamide imidazole ring
and one nitrogen in His-583 of eEF2 are the closest prospective nucleophilic atoms in eEF2
to the C1 nicotinamide ribose of the NAD+ substrate.
ACKNOWLEDGEMENTS
We thank Gerry Prentice for expert technical assistance during the course of these studies. This work
was funded by the Canadian Institutes of Health Research (to A.R.M.).
FIGURE LEGENDS
Figure 1: General reaction scheme for the ADPRT enzyme family. The reaction features
the SN1 attack by a nucleophilic atom (:Nu) of the amino acid of the target protein. In the
reaction scheme, the glycosidic bond is (step 1) broken followed by (step 2) the nucleophilic
attack by the incoming nucleophile of the target protein.
Figure 2. Sequence alignment of ADPRT family including the PARP group.
The DT group contains a histidine at position 4 from the left whereas the CT group contains
an arginine at position 4 from the left. The consensus sequences are -H-G-X…X-G-X-YX10Y-X…X-X-E… for the DT group and -R-X-X…X--X-S-T/E/A-S/Q/T…E/Q-X-E…
for the CT group. The numbers in brackets indicate the number of amino acids between given
sequences and the conserved residues are shown in bold text. The target residue for covalent
modification (ribosylation) is indicated in the far right column. The list is not meant to be
exhaustive but contains some representative members of each group. The motif consists of
three regions (regions 1- 3) and a beta strand separated by a beta strand-helix motif followed
by a single beta strand housing the essential catalytic Glu residue (beta, arrow; -helix,
shaded cylinder).
Figure 3. Core folded structures for ADPRT enzymes. The following structures were
taken from the RCSB Protein Structure Database: Pseudomonas aeruginosa exotoxin A
16
(1AER), diphtheria toxin (1TOX), chicken PARP (2PAW), pertussis toxin (1BCP), cholera
toxin (1XTC), E. coli heat-labile toxin (1LTA), exoenzyme C3 (1G24), and vegetative
insecticidal protein-2 (1QS1). Only the conserved core of the enzymes is shown and the
structures were positioned in a similar 3-D orientation. The colors correspond to the
following: red (catalytic Glu), pink (catalytic His in DT group or Arg in CT group), green
(conserved aromatic residue next to His or Arg in DT and CT groups, respectively), orange
(-strand with catalytic Glu), and blue (partner -strand to orange -strand).
Figure 4. Loop 3 residues. (A) Loop 3 of ETA in the crystal structure of the ETA-eEF2TAD complex (PDB entry, 1ZM4). The surface structure of ETA is shown in white and
the TAD in ball and stick format (black). Residues in Loop 3 are shown in ball and stick
representation and colored according to their importance for catalytic activity, where red is
the most important, orange residues are only weakly affected and white residues are
unaffected. The diphthamide of eEF2 is illustrated in green ball and stick mode. Blue bonds
indicate hydrogen bonds, whereas gray bonds only indicate the distance between Glu-546 and
the nucleophile at the diphthamide (DIPH) and the 2’ hydroxyl group on the ribose of TAD.
(B) Loop 3 in DT superimposed (PDB entry, 1DTP) on the crystal structure of the ETAeEF2-TAD complex. The surface structure of DT is shown in white and TAD (from the
ETA-eEF2-TAD complex) is rendered as black ball and sticks. Residues in Loop 3 of DT,
eEF2 and hydrogen bonds are shown as in A. (C) Sequence alignment of loop 3 in ETA and
DT. The alignment was completed by using LALIGN
(http://www.ch.embnet.org/software/LALIGNform.html)
Figure 5. Superposition of Loop 3 from various ETA X-ray structures. (A) Structural
alignment of Loop 3 motif in ETA complexes. (B) Structural alignment of Loop 3 motif
in various ADPRT enzymes.
17
Table 1: Relative ADPRT, GH and NAD+ Binding Activity for Pseudomonas aeruginosa Exotoxin A
Wild-Type and Mutant Proteins
Protein
a
Relative ADPRT
(kcat)
b
c
Wild-type
E546Ad
E547Ad
E548Ad
G549Ad
1.00  0.09
0.0012  0.00002
0.795  0.077
1.669  0.298
0.770  0.076
35  3
62  12 (1.8)
69  27 (2.0)
94  4 (2.7)
280  33
(8.0)
231  19
(6.6)
94  1 (2.7)
155  20
(4.4)
90  30 (2.6)
134  31
(3.8)
36  3 (1.0)
75  9 (2.1)
81  25 (2.3)
176  18
(5.0)
80  16 (2.3)
45  1 (1.3)
92  13 (2.6)
195  32
(5.6)
39  1 (1.1)
95  26 (2.7)
1.00  0.02
0.24  0.008
0.83  0.14
0.76  0.01
0.30  0.005
45  17 (1.3)
0.38  0.01
d
G550A
0.562  0.166
R551Ad
L552Ad
0.380  0.024
2.365  0.527
E553Ad
E546De
0.0015  0.00005
(2.7  1.0)10-4
E546He
E546Ne
E546Qe
R551Ee
0.0012  0.0002
0.0010  0.00009
0.011  0.002
0.011  0.005
R551He
R551Ke
R551Qe
R551Ce
g
0
0.317  0.024
0.185  0.014
0
E546A/R551Af 0.0001  0.0001
E546D/R551Kf 0.0009  0.0013
E546R/R551Ef
0.00001 
0.000002
Relative GH
(kcat)
KD (M)
Trp emission
maximum wavelength
(nm)
332
330
330
331
331
0.41  0.04
0.32  0.005
0.58  0.002
0.07  0.009
0.45  0.002
0.59  0.009
0.67  0.03
0.80  0.04
0.09  0.001
0.41  0.002
0.69  0.03
0.38  0.01
0.10  0.006
0.84  0.04
0.42  0.0002
333
331
331
331
332
331
331
331
331
332
332
331
332
331
331
331
a
The relative ADPRT activity was measured as described in Experimental Procedures and was set at
1.00 for the WT ETA enzyme (d746  18 min-1; e628  8 min-1; f847  21 min-1).
b
The NAD+ binding ability of WT and mutant ETA proteins was measured as described in
Experimental Procedures from the quenching of the intrinsic fluorescence of ETA by the binding of
NAD+. The numbers if parentheses represent the extent (fold) increase in KD compared with the WT
ETA.
c
The relative GH activity was measured as described in Experimental Procedures and was set at 1.00
for the WT ETA enzyme (d0.127 0.001 min-1; e0.129  0.002 min-1; f0.117  0.001 min-1).
g
The ADPRT activity could not be accurately measured since it was nearly zero.
The kinetic and equilibrium binding data represent the mean  S.D. from three independent
experiments.
18
Table 2: Comparison of the Loop 3 motif in some ADPRT enzymes
a
Protein
Loop
Length
Motif
H-bond
ETA
6
546 Glu N--O=C 551 Arg
DT
7
PARP
37
b-strand-loopb-strand
b-strand-loopb-strand
b-strand(loop-b-standloop)-b-strand
C3
11
exoenzyme
pertussis
29
toxin
cholera
toxin
15
vegetative 11
insecticidal
protein 2
iota-toxin
11
with
NADH
heat labile 15
toxin
b-strand-loopb-strand
b-strand(loop-helix)b-strand
b-strand(loop-helix)b-strand
b-strand(loop-helix)b-strand
b-stand-(loophelix)-b-stand
b-strand(helix-loop)b-strand
Type
of Hbond
main
chain
main
chain
main
chain
End
residues
of loop
Glu 546Arg 551
Phe 140Ser 146
Leu 951Asn 987
Critical
residue
main
chain
main
chain
Ile 203Leu 213
100 AlaSer 128
Glu 214
96 Asn O--N 111 Gln
side
chain
97 ValGln 111
Glu 112
417 Leu N-O=C 428 Glu
main
chain
417 Leu427 Lys
Glu 428
369 Leu N-O=C 380 Glu
main
chain
369 Leu379 Tyr
Glu 380
97 Val N--O=C 112 Glu
main
chain
97 ValGln 111
Glu 112
140 Phe N--O=C 146 Ser
977 Thr O--O=C 952 Gly
N--O=C 987 Asn
950 Gly N--O=C 988 Glu
C=O--N
203 Ile N--O=C 214 Glu;
204 Asp O--N Leu 213
100 Ala N—O=C 129 Glu
a
Glu 553
Glu 148
Glu 988
Glu 129
These proteins all possess high resolution X-ray crystal structures and loop regions were
rendered for proteins that do not have a substrate or ligand bound within the active site of the
enzymes. DS ViewerPro 5.0 was used to render the structures and to analyze the loop region,
including the identification of H-bonds within the structure. The proteins and their
corresponding RSCB PDB files are: ETA, 1IKQ; DTA, 1DTP; PARP, 2PAW; C3
exoenzyme, 1G24; pertussis toxin, 1PRT; cholera toxin, 1XTC; vegetative insecticidal
protein 2, 1QS1; iota-toxin with NADH, 1GIQ; heat labile toxin, 1LTS.
19
Table 3: Comparison of ADPRT activity, GH and NAD+ binding activity for wild-type and mutant
diphtheria toxin proteins
Protein
Wild-type DT
F140Hf
F140Pf
E142Ad
E142Dd
S144Af
S145Af
S146Ae
S146Te
S146Hf
S146Pf
a
Relative
ADPRT (kcat)
1.00  0.04
0.04  0.002
0.003  0.0005
0.59  0.15
0.79  0.04
0.71  0.06
0.81  0.04
0.79  0.04
0.58  0.03
0.26  0.02
0.02  0.001
b
c
KD (M)
11  1
31  4.5 (2.8)
28  9.4 (2.5)
32  0.4 (2.8)
32  12 (2.8)
26  2.0 (2.3)
29  7.0 (2.6)
34  4.7 (3.0)
44  3.9 (3.9)
40  1.6 (3.5)
60  5.8 (5.3)
a
Relative
GH (kcat)
1.00  0.15
0.51  0.12
0.43  0.10
0.96  0.08
1.40  0.18
0.84  0.03
1.04  0.09
0.87  0.04
0.55  0.14
0.65  0.14
0.45  0.13
Trp emission
maximum wavelength (nm)
332
332
332
332
332
332
332
332
332
332
332
The relative ADPRT activity was measured as described in Experimental Procedures and was set at
1.00 for the WT DTA enzyme (dkcat was 296  12 min-1; ekcat was 283  11 min-1; fkcat was 303  10
min-1).
b
The NAD+ binding ability of WT and mutant DTA proteins was measured as described in
Experimental Procedures from the quenching of the intrinsic fluorescence of DTA by the binding of
NAD+. The numbers in parentheses represent the extent (fold) increase in KD compared with the WT
DTA.
c
The wild-type kcat for GH activity was measured as described in Experimental Procedures and was
set at 1.00 for the WT DTA enzyme (0.0012  0.0002 min-1).
The kinetic and equilibrium binding data represent the mean  S.D. from three independent
experiments.
20
Figure 1
H2N
N
O
-phosphate
N
O
O
H C
P
P
CH2
O 2 O
O
O
O
O- ON
NAD+
N
-phosphate
A-ribose
HO
OH
H2N
STEP 1
N
-
NH2
N
H
OH
HO
O
+
N-ribose
NH2
N
nicotinamide
-phosphate
N
O
O
H2C
P
P
CH2
O
O
O
O
O
O- ON
N
+
-phosphate
HO
OH
H
HO
OH
oxacarbenium ion
A-ribose
STEP 2
H2N
:Nu
protein target
- H+
N
-phosphate
N
O
O
H2C
P
P CH2 O
O
O
O
O
O- ON
N
H
Nu
-phosphate
A-ribose
HO
OH
HO
OH
N-ribose
ADP-ribose--protein
21
protein target
Figure 2
ADPRT
Sequence
Region 1
1
DT Group
Diphtheria toxin
Exotoxin A
V. cholera putative ToxA
Chick PARP*
Human PARP-1
Human Vault PARP**
HumanTankyrase
CT Group
Pertussis toxin
C. botulinum C3 Exoenzyme
C. linosum ADPRT
S. aureus C3 Exoenzyme
Epidermal differentiation inhibitor
Vegetative insecticidal protein 2
S. enterica SpvB
Azosporillium sp. DRATab
Bacteriophage ADPRT
C. perfringes Iota-toxin
Human spleen ADPRT
Mouse Rt 6.1
Mouse lymphocyte ADPRT
Chick erythroblast ADPRT
Human skeletal muscle ADPRT
Rabbit skeletal muscle ADPRT
Mouse testes ADPRTb
Cholera toxin
E. coli Heat labile toxin
Exoenzyme S
Clostridium botulinum C2 toxin
Region 2
2
Helix 1
Target
residue
Region 3
3
XXXXФHGX…………XGXYXXXXXXXXXXYX…………XXXXXXXEXXXXXX (consensus)
SSYHGT(27)KGFYSTDNKYDAAGYS(74)AEGSSSVEYINNWE
DIPH
VGYHGT(24)RGFYIAGDPALAYGYA(62)EEEGGRLETILGWP
DIPH
VGYHGT(27)GGLYVATHAEVAHGYA(69)ESAGGEDETVIGWD
DIPH
LLWHGS(28)KGIYFADMVSKSANYC(72)DTCLLYNEYIVYDV
Glu
LLWHGS(28)KGIYFADMVSKSANYY(72)DTSLLYNEYIVYDI
Glu
PLLHGS(33)SGIYFSDSLSTSIKYS(50)TTDFEDDEFVVYKT
Glu
MLFHGS(23)AGIYFAENSSKSNQYV(59)VNGLAYAEYVIYRG
Glu
XXФRXX…………XФXSTSXXXXXXXXXX….……EXEXXXXXX
TVYRYD(37)AFVSTSSSRRYTEVYL(62)QSEYLAHRR
MLFRGD(40)GYISTSLMNVSQFAGR(26)QLEMLLPRH
ILFRGD(40)GYISTSLVNGSAFAGR(26)QLEVLLPRS
YVYRLL(47)GYSSTQLVSGAALAGR(27)QQEVLLPRG
YVYRLL(43)GYSSTQLVSGAAVGGR(26)QQEVLLPRG
TVYRWC(31)GYMSTSLSSERLAAFG(27)EKEILLDKD
VVYRGL(24)AFMSTSPDKAWINDTI(23)EAEMLFPPN
RLYRGV(34)DYILETWVPLTKVVFF(11)EGEVILPRG
TVYRAQ(19)NFVSTSLTPIIFGRFG(64)EAEVILPRG
IVYRRS(37)NFISTSIGSVNMSAFA(27)EYEVLLNHG
VHYRTK(15)QFLSTSLLKEEAQEFG(21)KKEVLIPPY
QVYRGV(17)GFASASLKNVAAQQFG(18)EEEVLIPPF
VVFRGV(17)QFTSSSVDERVARRFG(24)EREVLIPPH
NVFRGV(16)QFTSSSLQKKVAEFFG(23)EDEVLIPPF
QVFRGV(17)GFASASLKHVAAQQFG(18)EEEVLIPPF
QVFRGV(18)GFASASLKNVAAQQFG(18)EEEVLIPPF
SVYRGT(15)HFASSSLNRSVATSSP(27)EEEVLIPGY
KLYRAD(48)GYVSTSISLRSAHLVG(41)EQEVSALGG
RLYRAD(48)GYVSTSLSLRSAHLAG(41)EQEVSALGG
QTFRGT(18)GYLSTSLNPGVARSFG(23)EKEILYNKE
IAYRRV(43)SFSSTSLKSTPLSFSK(25)EQEILLNKN
a
(consensus)
Cys
Asp
Asp
Asp
Asp
Asp
Asp
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
E or Q is possible at this position, with one sequence, human spleen ADPRT, that possesses a K at
that position.
22
Figure 3
Pseudomonas aeruginosa Exotoxin A
Diphtheria toxin
Pertussis toxin
Cholera toxin
Chick PARP
E. coli Heat-labile toxin
Vegetative insecticidal protein-2
Exoenzyme C3
Fig. 6: Core folded structures for mono-ADPRT enzymes. The following structures were
taken from the RCSB Protein Structure Database: Pseudomonas aeruginosa Exotoxin A (1AER),
Diphtheria Toxin (1F0L, with NAD: 1TOX), Chick Poly ADPRT (2PAW), Pertussis Toxin
(1BCP), Cholera Toxin (1XTC), E. coli Heat-labile Toxin (1LTA), Exoenzyme C3 (1G24),
Vegetative Insecticidal Protein-2 (1QS1).
23
Figure 4
A
B
C.
546
ETA
DT
551 553
558
|
| |
|
PEEEGGR-LETILGW
: ::. .: : .:
PFAEGSSSVEYINNW
|
|
142
148
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