0
Structure of activated thrombin-activatable fibrinolysis inhibitor, a
molecular link between coagulation and fibrinolysis.
Laura Sanglas, 1,4 Zuzana Valnickova, 2,4 Joan L. Arolas, 3
Irantzu Pallarés, 1 Tibisay Guevara, 3 Maria Solà, 3 Torsten Kristensen, 2
Jan J. Enghild, 2 Francesc X. Aviles, 1,* & F. Xavier Gomis-Rüth 3,*
1
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Bio. Mol. (Facultat de
Ciències); Universitat Autònoma de Barcelona, E-08193 Bellaterra (Spain)
2
Center for Insoluble Protein Structures at the Department of Molecular Biology; Science Park;
University of Århus; Gustav Wieds Vej 10c, DK-8000 Århus C (Denmark)
3
Department of Structural Biology at the Molecular Biology Institute of Barcelona, CSIC; Barcelona
Science Park; c/ Balidiri Reixach,10-12 and 15-21; E-08028 (Spain)
4
These authors contributed equally to this study and share first authorship.
* Correspondence: <xgrcri@ibmb.csic.es> or <francescxavier.aviles@uab.es>. The authors state
they have no competing financial interest.
RUNNING TITLE: Active TAFI carboxypeptidase in complex with TCI
1
SUMMARY
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that
links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological
target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short
half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a
proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that
TAFIa conforms to the /-hydrolase fold of MCPs and displays two unique flexible loops on the
molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition,
point mutations reported to enhance protein stability in vivo are mainly located in the first loop and
in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts
both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency.
2
INTRODUCTION
After blood-vessel injury, hemostasis induces fibrin clot formation to prevent blood loss and
trigger vessel repair (Mann et al., 1988). This clot must be removed after tissue repair to restore
blood flow. These processes are tightly regulated by the coagulation and fibrinolytic cascades
because imbalance may lead to thrombosis, heart attack and stroke, or to bleeding as in hemophilia
(Boffa et al., 2001). Hemostasis is modulated by thrombin-activatable fibrinolysis inhibitor (TAFI),
also known as procarboxypeptidase (PCP) B from plasma, procarboxypeptidase B2, U and R. It
attenuates fibrinolysis by removing surface-exposed C-terminal lysine residues from the fibrin clot
(Arolas et al., 2007; Bajzar et al., 1995; Boffa et al., 2001; Bouma and Mosnier, 2003; Eaton et al.,
1991; Hendriks et al., 1989; Willemse and Hendriks, 2007). TAFI is the zymogen of a B-type zincdependent metallocarboxypeptidase (MCP), which is produced and secreted by the liver, and apart
from carboxypeptidase N, it is the only known MCP found in human plasma. TAFI is similar in
sequence to human pancreatic PCPs (see Fig. 1 and (Arolas et al., 2007), (Pereira et al., 2002)).
However, it differs from these proenzymes in that the pro-domain is highly glycosylated at four sites,
the glycosylation accounting for ~20% of the total molecular mass (Valnickova et al., 2006). During
coagulation, TAFI is processed by thrombin/thrombomodulin to TAFIa through removal of its 92residue pro-domain. In contrast to pancreatic MCPs, both TAFI and TAFIa uniquely display
carboxypeptidase activity against larger substrates. However, while TAFIa has a half-life of less than
ten minutes, in contrast to its robust pancreatic counterparts, TAFI is stable in circulation (Boffa et
al., 1998; Valnickova et al., 2007).
Protein inhibitors account for a control mechanism of proteolytic activity. Latexin, alias ECI,
is the only known endogenous A/B-type MCP protein inhibitor found in mammals (Pallarès et al.,
2005). Its role as a TAFIa inhibitor in fibrinolysis is questionable, however, as it is not found in
blood. In contrast, TCI is a physiologically relevant inhibitor that has been found in the
hematophagous ixodid tick, Rhipicephalus bursa, which only feeds on ruminants. Such parasites
need to inactivate host inflammation and defense mechanisms and prevent coagulation in the gut
3
during feeding through protein inhibitors. TCI displays the highest affinity for TAFIa (equilibrium
dissociation constants of 1.3 and 1.2 nM for bovine and human TAFIa, respectively) and strongly
accelerates fibrinolysis similarly to the A/B-type MCP inhibitors from potato (PCI) and leech (LCI),
which also target TAFIa. These protein inhibitors have proven potential as therapeutic adjuvants and
they are now under clinical trials in various cardiovascular conditions (Arolas et al., 2007). In
addition, stimulation of the TAFI pathway is being examined as an approach to the treatment of
hemophilia (Mosnier and Bouma, 2006; Walsh et al., 1971). Such research attests to the importance
of TAFI as a pharmacological target for cardiovascular disease treatment (Do et al., 2005; Hsu et al.,
2007; Klement et al., 1999; Refino et al., 2000). Recent data show that the TAFI plasma
concentration in humans varies strongly and that high concentrations are a risk factor for thrombosis
and coronary artery disease (Silveira et al., 2000; van Tilburg et al., 2000), while low levels have
been correlated with chronic liver disease (Van Thiel et al., 2001). Accordingly, TAFI has been
proposed as a molecular marker for vascular diseases (Boffa and Koschinsky, 2007; Boffa et al.,
2001; Mann et al., 1988; Wang et al., 2007).
In addition to its involvement in fibrinolysis, TAFI has been implicated in wound healing,
blood-pressure regulation, tissue remodeling and inflammation by inactivating plasminogen
activation, bradykinin and inflammatory mediators (for a review, see (Arolas et al., 2007)), as well as
in sepsis (Taylor and Bajzar, 2005), endometriosis (Bedaiwy and Casper, 2006), and pulmonary
fibrosis (Gabazza et al., 2006). TAFI is also expressed in the neuronal endoplasmic reticulum of
hippocampal neurons, potentially playing a role in the processing of native -amyloid precursor
protein in the brain (Matsumoto et al., 2000; Matsumoto et al., 2001). Here, latexin (alias ECI),
which has been detected in rodent and human brain (Arimatsu, 1994; Normant et al., 1995), could
play a role as an endogenous TAFI inhibitor.
Given the importance and key role of the TAFI/TAFIa axis in thrombotic conditions, the
detailed structure analysis of TAFIa should contribute to an explanation for its intrinsic instability
and provide a mold for the design of small-molecule inhibitors, thus contributing to alternative
therapeutic approaches.
4
RESULTS AND DISCUSSION
TAFI purification, complex formation with TCI and heparin-binding assay – Glycaninduced heterogeneity, the minute amounts retrievable from single-patient samples, the impossibility
of establishing efficient recombinant over-expression systems, and the instability of human TAFIa
have hampered structural studies on this protein since its discovery in 1988. We sought to overcome
these problems by choosing a highly homologous orthologue from a non-human mammalian species,
for which large amounts of blood could be obtained from one individual. We purified TAFI from a
single cow to homogeneity, essentially in three large-scale liquid chromatography steps, and showed
it to be active against a chromogenic substrate (see materials and Methods). The kinetic properties of
bovine TAFI and human TAFI were compared in parallel experiments and the data suggested that the
two proteins are very similar in terms of activity (Table 1), in agreement with their 78% sequence
identity (see Fig. 1). Freshly purified TAFI was subsequently incubated in vitro with the
physiological processor thrombin in the presence of its modulator thrombomodulin and TCI. The
TAFIa/TCI complex was stable for several days/weeks and suitable for structural studies. In addition,
our experiments with heparin-sepharose showed that both human and bovine TAFI binds heparin,
since most of the protein eluted only in the presence of 200-500mM NaCl (data not shown). This
suggests that glycosaminoglycans may modulate TAFI as described for antithrombin III and
thrombin (Bjork and Lindahl, 1982).
Overall structure of TAFIa, active-site cleft and potential heparin-binding sites - TAFIa has
a compact globular shape and shows the classic /-hydrolase fold of A/B- and N/E-type MCPs
(Arolas et al., 2007; Vendrell et al., 2000). It has a central eight-stranded -sheet (strands 1-8) of
strand connectivity +1,+2,-1x,-2x,-2,+1x,-2, which vertically spans the molecule top to bottom
accumulating a vertical twist of ~130º (Figs. 1 and 2a). This gives rise to a concave face, which
accommodates helices 4 and 6, and the active-site cleft. At the rear, the convex side of the sheet
harbors 1-3, 5 and 7 and the surface N- and C-termini of the molecule. The access to the active
site is like a funnel, whose rim is shaped by a series of irregular loop segments required for
5
interactions of the protease moiety with the pro-domain and with cognate protein inhibitors in A/Btype MCPs (Pallarès et al., 2005). These segments include the loop connecting strand 8 with helix
7 (L87), L32, L56, L65, as well as the first part of the 53-residue segment connecting
3 and 4 (L34). This long segment is stabilized by two disulfide bonds (Cys138-Cys161 and
Cys152-Cys166; for numbering conventions, see Fig. 1) and closes the front and the bottom of the
active site and the specificity pocket, thus contributing to the characteristic cul-de-sac of these
exopeptidases (Fig. 2c).
The catalytic zinc ion resides at the bottom of the funnel-like cleft and is coordinated by His69
N1 (2.09Å apart) and by Glu72, in an asymmetric bidentate manner, through its O2 (2.01Å) and
O2 (2.76Å) atoms (Fig. 2c). These two residues are imbedded in a consensus sequence, HXXE
(amino-acid one-letter code; X for any residue), characteristic of A/B- and N/E-type MCPs (Hooper,
1994). The third TAFIa zinc ligand is His196 N1 (2.08Å). An acetate ion is found next to the zinc,
partially occupying the specificity pocket and hydrogen-bonded through one of its carboxylate
oxygen atoms to Arg127 N2 (3.02Å), Arg145 N2 (2.89Å), and Tyr248 O (2.58Å). The latter is
in the “down” orientation usually found in MCPs with occupied pockets (Reverter et al., 2000). The
other acetate carboxylate oxygen atom is bound to Asn144 N2 (2.90Å) and Arg145 N1 (2.80Å).
As shown for other MCPs, the protein residues engaged in substrate binding and catalysis in A/Btype MCPs (Auld, 2004; Vendrell et al., 2000) are Arg127 and Glu270 forming S1; Arg71, Ser197,
Tyr198, and Ser199 delimiting S2; and Phe279 contributing to S3. Typical B-type MCP specificity
towards basic side chains in substrates is due to an S1’ pocket that is hydrophobic at its entrance and
acidic at its bottom. The pocket is formed by the side chains of Asn144, Arg145, Val203, Gly243,
Leu247, Ala250, Thr268, Tyr248, and Asp255. The terminal carboxylate group of a substrate, when
it is trapped for scission, is fixed by Asn144, Arg145, and Tyr248, while the scissile carbonyl group
is near Glu270, Arg127, and the catalytic zinc (Fig. 2c).
Heparin has been shown to stabilize TAFIa against spontaneous inactivation (Mao et al.,
1999). In the absence of structural data of a complex with heparin, surface anions may point to
6
potential binding sites. In the present complex, two pairs of sulfate ions found in the structure may
represent two potential independent binding sites for heparin sulfate groups. In one case, the two
sulfate ions are 10Å apart, one is bound by Ser158 N and O, and Arg52I N1 and N2 from the
TCI moiety, and the other is bound by Ser160 N and O, and Ala137 N. The distance between sulfate
ions is similar to that found in the thrombin/heparin complex structure, in which two sets of sulfate
groups from sulfoiduronate or disulfoglucosamine moieties are separated by two (11Å apart) and
three (10Å apart) monosaccharides, respectively (Protein Data Bank (PDB) access code 1XMN;
(Carter et al., 2005)). The sulfur atoms of the second potential TAFIa site are 20Å apart, which is
compatible with the spacing of three or four monosaccharides within a heparin chain (Carter et al.,
2005). Here, the sulfate groups are bound by His1501 N2, Ser209 O, and Ser211 O and by His216
N2, Arg224 N2 and N, and Ser 220 O, respectively, and they affect segment 5-L57-7 on
top of the molecule (Fig. 2a). It is interesting to note that this region contains a high number of
residues whose substitution by random mutagenesis has been shown to influence TAFIa stability
((Knecht et al., 2006; Schneider et al., 2002); see Fig. 1 and below). Therefore, heparin binding at
this site could thus become a regulatory mechanism affecting the half-life of TAFIa.
For the last six years, the structure of human pancreatic PCPB1, solved by two of our groups,
has been a model for TAFI (Marx et al., 2000; Pereira et al., 2002). Sequence and structure
comparison of the homology model obtained from the former with bovine TAFIa (Fig. 1 and 2b)
enables to assess that the gross of the model proposed was valid. In particular, TAFI very likely
possesses an equivalent pro-domain to PCPB1 with a globular part folded as an  open sandwich.
This globular part would be linked to the mature enzyme moiety through a connecting segment,
which would include an -helix. The pro-domain would be likewise placed on top of the active site,
which would be preformed in the TAFI zymogen. With respect to the mature enzyme moiety, the
overall structure is also very similar and this similarity comprises the identity and arrangement of the
active-site residues (see also below). There are two major points that were anticipated by the PCPB1based model: (i) An essential salt bridge made up between Asp41 of the pro-domain and by an
7
active-site residue, Arg145, is not present in TAFI as inferred from TAFIa. This interaction is
thought to account for the lack of activity of B-type MCP zymogens and therefore it was postulated
that TAFI might show intrinsic activity, at least against small substrates (Pereira et al., 2002). This
hypothesis was recently proven right for experiments in vitro (Valnickova et al., 2007). (ii) Our
model further anticipated that L24 could be a hot spot accounting for instability as it showed
highly-positive values of pseudo-potential energy (Pereira et al., 2002). This is confirmed by the
present study (see below).
Structural determinants of TAFIa inhibition through TCI – The proteinaceous inhibitor TCI
consists of tandem structurally-similar small modules, an N-terminal (NTD; Asn1I-Leu37I) and a Cterminal domain (CTD; Cys40I-Leu74I) linked by two residues, Thr38I and Gly39I (Fig. 2a). Each
domain is compacted by three internal disulfide bonds and consists of a short -helical fragment and
a subsequent antiparallel triple-stranded -sheet. This fold resembles -defensin, as previously
reported (Arolas et al., 2005b). The structural similarity of the NTD and the CTD suggests that this
inhibitor may have arisen by gene duplication. The two TCI domains do not interact with each other
suggesting they are flexible in solution. This feature means that the two domains could bind
simultaneously to separate sites of a target protease, which would provide for more potent and
selective inhibition.
The way in which TCI inhibits TAFIa resembles its inhibition of human CPB1 (PDB 1ZLI;
(Arolas et al., 2005b)). Both complexes display an rmsd of 0.76Å for 368 common C atoms
deviating by less than 3Å. TCI mainly inhibits TAFIa through its CTD (Fig. 2c), with an interaction
surface between the enzyme and the inhibitor spanning 666Å2. This domain lies on top of the funnellike rim surrounding the active-site cleft and establishes 32 contacts (<4Å) with the TAFIa protein
moiety. As observed in other MCP/inhibitor complexes, such as those made by PCI (Rees and
Lipscomb, 1982) and LCI (Reverter et al., 2000), the C-terminal residue, here His75I, is cleaved and
the new C-terminus, Leu74I, approaches and contacts the TAFIa catalytic zinc ion through its
carboxylate oxygen atoms (2.12 and 2.52 Å apart). This means that the metal ion is coordinated by a
total of six ligands in a basically tetrahedral arrangement (two oxygen and two nitrogen atoms 2.01-
8
2.12Å apart), with another two oxygen ligands at 2.52 and 2.76Å (Fig. 2c). In addition to this
contact, CTD interacts with TAFIa through 15 hydrogen bonds and five hydrophobic contacts (see
Table 2). The NTD of TCI contacts TAFIa over a surface of 413Å2 and mainly interacts with TAFIa
region Trp120-Met125 at the beginning of the long L34 segment. This interaction includes 21
contacts (<4Å), among them six hydrophobic contacts and seven hydrogen bonds and polar
interactions (see Table 2). Accordingly, the interaction mediated by NTD is weaker than that
performed by CTD, but it identifies Trp120-Met125 as an exosite contributing to complex stability,
which could be targeted by inhibitors in addition to the active-site to enhance specificity and potency
(Fig. 2d). This exosite is actually specific for the dual binding mode of the inhibitor as it was also
found in the complex of TCI with human CPB (Arolas et al., 2005b). Interestingly, although the
composite complex interaction surface provided by NTD and CTD (1,080Å2) is smaller than typical
protease-inhibitor interfaces (1,250-1,750Å2, (Janin and Chothia, 1990), the number and type of
contacts in the TCI/TAFIa complex renders it as stable as that formed by human CPA4 and latexin,
which displays a much larger contact surface (1170Å2) but fewer intermolecular contacts and no
direct interaction with the active-site cleft (Pallarès et al., 2005).
Structural determinants of TAFIa instability, conformational rearrangement and proteolytic
inactivation in vivo –The short half-life of TAFIa has been correlated with a temperature-dependent
conformational instability, which renders the molecule dysfunctional and eventually increases its
susceptibility to proteolysis (Arolas et al., 2007; Boffa et al., 2000; Boffa et al., 1998). Several
TAFIa variants have lower thermal sensitivity and thus longer half-lives and a greater antifibrinolytic
potential (Ceresa et al., 2007; Ceresa et al., 2006; Marx et al., 2004). These variants include point
mutants (Fig. 1), as well as two chimeric constructs between human TAFI and CPB1 containing
segments 201-240 and 201-308 from the latter, respectively. Inspection of the respective regions in
the TAFIa structure shows that these mutations map onto structure elements L23, L34, 6,
L65, 5, L57, 7 and 6. The beneficial mutations accumulate at segment 5-L57-7,
which lies on top of the molecule between Arg210 and Ser242 (Figs. 1 and 2a). In addition, residues
in this segment, Arg210, Lys235 and Arg237, have been identified as hot spots for proteolytic
9
inactivation of TAFIa through thrombin and plasmin (Boffa et al., 2000; Boffa et al., 2001). This
segment is fully defined and rigid in the TAFIa structure and it is topologically equivalent in the
stable A/B-type MCPs such as human CPB1. However, its co-localization with a putative heparinbinding site (see above), together with the experimental evidence that human TAFI may bind
heparin, suggests a role for segment 5-L57-7 in the regulation of TAFIa half-life.
The present TAFIa structure provides evidence for another hypothesis for the mechanism of
destabilization, which is supported by stronger evidence: Two vicinal segments contained in L23
and L24 are flexible in the TAFIa structure and five residues are untraceable within each loop
(Figs. 1 and 2b). Intrinsically flexible regions on the molecular surface of a protein structure can be
directly correlated with instability, conformational changes/lability and proteolytic susceptibility,
and, thus, degradation (Zappacosta et al., 1996), especially if we consider a protease-rich medium
like blood. The equivalent regions of all other A/B-type MCPs thus far structurally analyzed are well
ordered and rigid, inter alia in active and zymogenic human pancreatic CPB1 (except for some
isolated side chains; see Fig. 2b and (Arolas et al., 2005b; Pereira et al., 2002)). Flexibility may be
favored in TAFIa by a one-residue insertion after position 56, which is absent in all pancreatic MCPs
(see Fig. 1 and (Pereira et al., 2002)). In addition, a unique lysine potentially targetable by thrombin
and plasmin is found at position 55 within L23 both in bovine, human, mouse and rat TAFIa, but
it is absent in pancreatic counterparts (Fig. 1). In contrast to the preceding lysine at position 54,
Lys55 should protrude from the TAFI molecular surface and be accessible to the action of proteases.
Mutation of Lys55 to asparagine has actually been found in human TAFIa variants that have 2.6times (a double point mutant) and 22-times (a fourfold mutant) longer half-lives than the wild-type
enzyme (Knecht et al., 2006). Furthermore, mouse, rat and bovine TAFIa undergo cleavage within
L23 as part of their inactivation processes. These findings back comparative studies of human and
rodent orthologues, which revealed similar but not identical biochemical characteristics, thus
suggesting a similar role during fibrinolysis in vivo ((Hillmayer et al., 2006) and our unpublished
results). According to the preceding considerations, the two-loop flexible region here described
could be conceived as hot spots to trigger destabilization and inactivation of the entire molecule.
10
In summary, the 3D crystal structure of TAFIa shows unique flexible features, which account
for the instability of the molecule as compared to the robust pancreatic counterparts. In the absence
of an endogenous inhibitor in the blood stream, it must be assumed that structural instability is the
principal modulator of TAFI/TAFIa activity, as was revealed by fluorescence studies (Boffa et al.,
2000; Ceresa et al., 2007). Major structural rearrangement leads to a loss of enzymatic activity,
which in turn causes sites susceptible to proteolysis to become available for degradation (Boffa et al.,
2000; Marx et al., 2000). In addition, the structural determinants of inhibition through a protein
inhibitor revealed by the present study may pave the way for the design of TAFIa inhibitors to be
used in thrombolytic therapies. The discovery of an exosite provides additional elements to be
considered for drug design. Initial steps in this direction have already been undertaken: TCI markedly
accelerates lysis of human plasma clots and its usage as an adjuvant is a promising approach (Arolas
et al., 2005b; Arolas et al., 2007).
11
MATERIALS AND METHODS
Preparation of human and bovine TAFI – Human TAFI was purified as described (Eaton et
al., 1991; Wiman, 1980) and its functionality was tested as previously reported (Valnickova et al.,
2007). Bovine TAFI was purified from 10L of bovine blood collected at the local slaughterhouse
from a single cow using 10mM EDTA as anticoagulant. At this concentration, the chelating agent
has no influence on the activity, i.e. the zinc ion is not removed. Plasma was recovered by
centrifugation and polyethylene glycol (PEG) 8000 was then added to a final concentration of 6%
(w/v). After 1h, the precipitate was collected by centrifugation and discarded. The supernatant was
applied to a 1L ECH-lysine-sepharose column (GE Healthcare), equilibrated in binding buffer
(50mM NaH2PO4, 100mM NaCl, pH7.5). The plasminogen-depleted flow-through was applied to a
500mL-plasminogen-sepharose column equilibrated with binding buffer. The column was washed
and bovine TAFI was eluted using 50mM -amino-caproic acid in binding buffer. After a buffer
exchange to 20mM Tris·HCl, pH7.5 (buffer B), bovine TAFI was separated from contaminating
proteins by using a GE Healthcare 5mL-HiTrap Q-HP anionic-exchange chromatography column
connected to an ÄKTAprime plus system (GE Healthcare). The column was developed with a linear
gradient from 20mM Tris·HCl, pH7.5 (buffer A) to 20mM Tris·HCl, 1M NaCl, pH7.5 (buffer B) at a
flow-rate of 3mL/min and a 0.5% B increase/min.
Determination of activity – Human and bovine TAFI activity was essentially determined as
described previously (Buelens et al., 2008). Briefly, 1µg of the zymogen was incubated with
increasing concentrations of the Hippuryl-Arg substrate (0-30mM), in duplicates, for 60min at 37C
in a final volume of 60µl. The reactions were terminated by the addition of 20µl 1M HCl followed
by 20µl of 1M NaOH and 25µl of 1M NaH2PO4, pH 7.4. Upon addition of 60µl 6% cyanuric
chloride dissolved in 1,4-dioxane, the samples were vortexed vigorously and centrifuged at 16000g
for 5min. The supernatant was subsequently transferred to 96-well microtiter plate and the
absorbance measured at 405nm in a FLUOStar Omega plate reader (BMG Labtech) using the
endpoint mode.
12
Heparin binding assay of human TAFI – Human TAFI (15g) in 500L of buffer A, was
incubated with 100L of heparin-sepharose for 1h at 22ºC. The supernatant was removed and TAFIbound heparin-sepharose was washed in 5x1mL buffer A. Elution of TAFI proceeded in five steps of
100L buffer A containing increasing amounts of NaCl.
TAFIa/TCI complex formation and purification – Recombinant TCI inhibitor was expressed
and purified as previously published (Arolas et al., 2005a). Bovine TAFI was activated by incubating
1mg-batches of zymogen (0.1mg/mL in 20mM Tris-HCl, 200mM NaCl, pH7.5) with 10µg of rabbit
thrombomodulin (American Diagnostica), 5µg of human thrombin (Sigma) and 0.4mg of TCI for 2h
at 25ºC. The resulting TAFIa/TCI complex was immediately purified by hydrophobic interaction
chromatography on a Resource Phenyl column (GE Healthcare) eluting with a decreasing linear
gradient from 1M (NH4)2SO4 to 0 in buffer B (50mM Tris·HCl, 150mM NaCl, pH7.5). Purified
complex obtained from several batches was pooled and injected to a HiLoad Superdex 200 26/60
column (GE Healthcare) equilibrated with buffer B. The complex was concentrated and bufferexchanged to 10mM Tris·HCl, 50mM NaCl, pH7.5 using an Amicon Ultra-4 concentrator (5kDacutoff, Millipore) and subsequently an Amicon Centricon concentrator (10kDa-cutoff, Millipore) to a
final concentration of 8.5mg/mL. The purity and integrity of the complex were assessed by SDSPAGE and mass spectrometry (data not shown).
Crystallization of the complex - Crystallization assays were performed following the sittingdrop vapor diffusion method. Reservoir solutions were prepared by a Tecan robot and 200-nL
crystallization drops were dispensed on 96x3-well CrystalQuick plates (Greiner) by a Cartesian
nanodrop robot (Genomic Solutions) at the joint IBMB-CSIC/IRB/Barcelona Science Park HighThroughput Crystallography Platform (PAC). Best crystals appeared after 3-4 days in a Bruker
steady-temperature crystal farm at 20°C with 0.2M (NH4)2SO4, 0.1M NaAcO, 10% PEG 4000,
pH4.6 as reservoir solution. These conditions were efficiently scaled up to the microliter range with
Cryschem crystallization dishes (Hampton Research). A complete diffraction dataset was collected at
100K from a single N2 flash-cryo-cooled (Oxford Cryosystems) crystal on a marCCD detector at
beam line ID23-2 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) within
13
the Block Allocation Group "BAG Barcelona". Crystals were trigonal and harbored one complex per
asymmetric unit. Diffraction data were integrated, scaled, merged, and reduced with programs
MOSFLM and SCALA within the CCP4 suite (CCP4, 1994) (see Table 3).
Structure solution and refinement - The structure was solved by Patterson-search methods
with program AMoRe (Navaza, 1994) using all diffraction data between 15 and 4Å resolution. The
coordinates of the complex between human CPB1 and TCI (PDB 1ZLI) were used as a searching
model. A single solution was found at 55.9, 47.1, 288.0 (,, in Eulerian angles) and 0.339, 0.319,
0.337 (x,y,z, as fractional unit-cell coordinates) after rigid-body refinement. This solution gave a
correlation coefficient in structure factor amplitudes of 51.1% and a crystallographic Rfactor of 42.9%
(for definitions, see Table 3 and (Navaza, 1994)). Subsequently, manual model building on a SiliconGraphics workstation using program TURBO-Frodo alternated with crystallographic refinement with
REFMAC5 within the CCP4 suite until completion of the model (see Table 3). This model contained
the protein residues of the mature protease moiety (molecule A) from Ser7 to Val308 (see Fig. 1 for
the numbering convention) and the catalytic zinc ion. Two segments, Lys55-Ala58 and Glu93-Thr97
were undefined and thus not included in the final model. The TCI model (molecule B; suffix I) was
fully defined in the complex for its 74 residues (Asn1I-Leu74I, after cleavage of the C-terminal
His75I residue).
Miscellaneous – Figure 2 was prepared with program MOLMOL (Koradi et al., 1996). The
final coordinates of the TAFIa/TCI complex have been deposited with the PDB at www.pdb.org
(access code 3D4U).
14
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ACKNOWLEDGMENTS
This study was supported by the following grants: BIO2007-68046, BIO2006-02668,
BFU2006-09593, PSE-010000-2007-1, and the CONSOLIDER-INGENIO 2010 Project “La Factoría
de Cristalización” (CSD2006-00015) from Spanish ministries; and 2005SGR00280 and
2005SGR01027 from the Generalitat de Catalunya. Additional funding was obtained by J.J.E. from
the Danish National Science Research Council. L.S. and I.P. enjoyed Ph.D.-fellowships from the
Spanish Ministry of Education and Science. M.S. and J.L.A. are, respectively, beneficiaries of the
“Ramón y Cajal” and “Juan de la Cierva” Programs of the Spanish Ministry of Education and
Science. We acknowledge the help provided by EMBL and ESRF synchrotron local contacts.
Funding was provided by ESRF for data collection. Robin Rycroft and, specially, Josep Vendrell are
thanked for helpful suggestions to the manuscript.
19
FIGURE LEGENDS
Figure 1. Alignment of B-type MCPs. Sequence alignment of bovine, human, rat and mouse
TAFI, human PCPB1 and porcine PCPB. The corresponding UniProt sequence database access
codes and the percentage of sequence identity with human TAFI within overlapping residues are
shown preceding the second and third blocks of sequences, respectively. Signal peptides are shown
over yellow background. The TAFI activation cleavage site is indicated by red scissors. The
traditional sequential numbering and the structure-based numbering of TAFI employed throughout
the text are shown above and below each alignment block, respectively. The latter numbering was
established for porcine PCPB to fit that of archetypal bovine CPA1 (Coll et al., 1991). This entails
that the pro-domain is numbered separately (1A-95A) from the active enzyme moiety (4-308) and
that there is a one-residue insertion after position 188 in B-type MCPs, here named 1889. TAFI starts
with Phe7A and has three extra unique one-residue insertions after positions 56, 150 and 235 of the
mature enzyme moiety with respect to porcine PCPB, which are numbered 567, 1501 and 2356,
respectively. In addition, TAFI displays three extra residues at the end of the pro-domain, so that
cleavage occurs at bond Arg98A-Ala4. Regular secondary structure elements for bovine TAFIa
(helices as green cringles, 1-7; -strands as red arrows, 1-8; T1 and T2 for 1,4-turns of type I
and II) are depicted above each sequence block. Identical residues in bovine and human TAFI are
shown over blue background and those identical in all four sequences over magenta background.
Positions reported to produce human TAFI or TAFIa variants with longer half-lives (Ceresa et al.,
2007; Ceresa et al., 2006; Knecht et al., 2006; Schneider et al., 2002) are shown in green. Segments
disordered in TAFIa are shown in red.
Figure 2. Structure of TAFIa in its complex with TCI. (A) Richardson plot of the complex
showing TAFIa in standard orientation with yellow -strands (1-8), green -helices (1-7) and
the catalytic zinc ion as a red sphere. Segment 5-L57-7 is shown in orange and the TAFI
exosite in magenta. The region of the proposed fibrinolysis switch is shown over gray background.
TCI is shown for its NTD (light blue), linker (red) and CTD (navy blue). The disulfide bonds of TCI
20
are also shown. The N- and C-termini of both molecules are labeled. (B) Close-up view of (A) after
a vertical rotation of ~90º showing the proposed fibrinolysis switch region of TAFIa (yellow)
superimposed with the equivalent region of human CPB1 (green). TAFIa regular secondary structure
elements are labeled. (C) Close-up view of (A) showing the TAFIa active site and the residues
participating in the interaction with TCI CTD. (D) Close-up view of (A) centered on the interaction
area of TAFIa with TCI NTD and the participating residues.
21
Table 1. Comparison of the activity of human and bovine TAFI.
human TAFI
Equation
bovine TAFI
Vmax
Km
Vmax
Km
(µM/min)
(mM)
(µM/min)
(mM)
Hanes
44.84
2.36
47.40
3.58
Eadie-Hofstee
44.65
2.35
44.33
3.14
Eisenthal-CornishBowden
44.96
2.44
44.61
3.16
44.99±1.19
2.35±0.20
47.84±3.67
3.88±0.85
44.86
2.38
46.05
3.44
Hyperbolic Regression
Average values
Kcat
(min-1)
Kcat/Km
(min-1/mM)
160.21
164.46
67.32
47.81
The values for Km and Vmax were determined using the direct fit of the Michaelis-Menten equation
employing 4 graphical methods. The data represent the enzyme-catalyzed reaction for 0.28µM TAFI.
22
Table 2. Interactions between TCI NTD and CTD with bovine TAFIa.
CTD
TAFIa
Dist.(Å)
Hydrogen bonds and polar interactions
NTD
TAFIa
Dist.(Å)
Hydrogen bonds and polar interactions
Gly44I O
Arg71 N1
3.40
Asn1I N2
Trp120 O
2.94
Glu46I O1
Glu163 N
2.77
Cys10I S
Lys121 O
3.17
Gln53I N2
Ser244 O
3.34
Cys10I O
Arg124 N
2.76
Gln53I O1
Leu249 N
2.87
Ser28I N
Lys121 O
2.78
Lys55I N
Ser246 O
3.28
Pro12I N
Lys122 O
3.36
Lys55I N
Leu247 O
3.04
Gly26I O
Lys121 N
3.12
Trp73I O
Arg71 N2
2.98
Thr29I O
Lys122 O
2.77
Trp73I O
Arg127 N1
3.29
Residues making van-der-Waals interactions
Leu74I O
His69 N1
3.10
Val4I
Trp73
Leu74I O
Glu72 O2
2.92
Val4I
Trp120
Leu74I O
Arg127 N1
2.83
Pro12I
Asp123
Leu74I OT
His196 N1
3.30
Ser28I
Lys122
Leu74I OT
Glu270 O2
2.59
Leu34I
Leu280
Leu74I OT
Ser197 O
3.26
Leu34I
Met125
Leu74I N
Tyr248 O
2.88
Residues making van-der-Waals interactions
Gly44I
Met125
Leu74I
Leu247
Val72I
Phe279
Trp73I
Ile164
Trp73I
Tyr248
23
Table 3. Crystallographic data.
____________________________________________________________________________
Space group / cell constants (a and c, in Å)
P3221 / 84.20, 128.90
Wavelength (Å)
0.8726
No. of measurements / unique reflections
Resolution range (Å) (outermost shell)a
874,530 / 57,962
48.1 – 1.70 (1.79 – 1.70)
Completeness (%)
Rr.i.m. (= Rmeas) b / Rp.i.m.
93.3 (95.5)
b
0.099 (0.611) / 0.025 (0.190)
Average intensity (<[<I> / (<I>)]>)
20.6 (4.0)
B-Factor (Wilson) (Å2) / Average multiplicity
18.6 / 15.1 (9.2)
Resolution range used for refinement (Å)
48.1 – 1.70
No. of reflections used (test set)
57,265 (695)
Crystallographic Rfactor (free Rfactor) c
0.156 (0.171)
d
No. of protein atoms / solvent molecules /
ions
2,991 / 358 /
1 (Zn2+), 2 acetates, 5 sulfates
Rmsd from target values
bonds (Å) / angles (°)
bonded B-factors (main chain / side chain) (Å2)
Average B-factors for protein atoms (Å2)
0.015 / 1.35
0.96 / 2.30
19.6
Main-chain conformational angle analysis
Residues in favored regions / outliers / all residues
351 / 1 / 362
_____________________________________________________________________________
a
Values in parentheses refer to the outermost resolution shell.
Rr.i.m.= hkl(nhkl /[nhkl-1] 1/2)i |Ii(hkl) - <I(hkl)>| / hkli Ii(hkl) and Rp.i.m.= hkl(1/[nhkl-1]1/2)i |Ii(hkl)
- <I(hkl)>| / hkli Ii(hkl).
b
c
Crystallographic Rfactor = hkl ||Fobs| - k |Fcalc|| / hkl |Fobs|; free Rfactor, same for a test set of reflections
not used during refinement.
d
Including atoms in alternate conformation.