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3D-modeling of thrombin-fibrinogen interaction*
Thierry Rose and Enrico Di Cera§
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St.
Louis, Missouri, 63110.
*This work was supported in part by the National Institutes of Health Research Grants HL49413 and
HL58141.
§To whom correspondence should be addressed: Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine, Box 8231, St. Louis, MO 63110; Tel: (314) 362-4185, Fax:
(314) 747-5354, e-mail: [email protected]
Running Title: Thrombin-fibrinogen interaction
Keywords: thrombin, fibrinogen, molecular modeling
Abbreviations used: FpA, fibrinopeptide A; FpB, fibrinopeptide B; PPACK, H-D-Phe-Pro-Arg-CH2Cl.
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on March 18, 2002 as Manuscript M110977200 by guest on February 16, 2018
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Abstract: 3D-models of thrombin complexed with large fragments of the fibrinogen Aα and Bβ chains
are presented. The models are consistent with the results of recent mutagenesis studies of thrombin and
with the available information on naturally occurring fibrinogen mutants. Thrombin recognizes fibrinogen
with an extended binding surface, key elements of which are Y76 in exosite I, located about 20 Å away
from the active site, and the aryl binding site located in close proximity to the catalytic triad. A highly
conserved aromatic-Pro-aromatic triplet motif is identified in the primed site region of fibrinogen and
other natural substrates of thrombin. The role of this triplet, based on the 3D-models, is to correctly
orient the substrate for optimal bridge-binding to exosite I and the active site. The 3D-models suggest a
possible pattern of recognition by thrombin that applies generally to other natural substrates.
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Thrombin plays multiple functional roles in the blood by interacting with a variety of proteins. The
procoagulant role of thrombin unfolds upon interaction with fibrinogen and culminates in the formation of
fibrin intermediates that polymerize into a blood clot. The kinetic steps defining this interaction and the
mechanism of fibrin polymerization have been characterized in great detail (1). The determinants of
fibrinogen recognition by thrombin have also been elucidated by site-directed mutagenesis (2,3) and
provide an important database of information that complements clinical findings on naturally occurring
fibrinogen variants that are associated with bleeding phenotypes.
The thrombin-fibrinogen interaction has enjoyed renewed interest in recent years after the
landmark solution of the crystal structure of fibrinogen derivatives (4-7). These structures have offered
clues on the molecular events underlying the end-to-end assembly of fibrin monomers generated after
the thrombin induced release of FpA from the Aα chain, the subsequent formation of fibrin protofibrils
and the thrombin induced release of FpB from the Bβ chain (4). Information on the structural epitopes of
thrombin recognizing fibrinogen has also been obtained. Fragments of FpA covalently bound to the active
site S195 of thrombin have been reported by X-ray crystallography (8,9) and NMR spectroscopy (10).
These studies have delineated the interactions made by FpA with the active site moiety of thrombin.
It has long been known, however, that regions of thrombin away from the active site are
involved in fibrinogen recognition (11). Specifically, exosite I located about 20 Å away from the active site
contains residues that are critical for fibrinogen binding. Macromolecular bridge-binding of fibrinogen to
exosite I and the active site confers high specificity to the interaction and serves as a binding mode
exploited by other natural substrates, like the G-protein coupled thrombin receptors (3). Recent Ala
scanning mutagenesis studies of thrombin have identified a hot spot in exosite I, centered around Y76,
that stores much of the energy responsible for the interaction with fibrinogen (2,3). Unfortunately, the
available crystal structure of the thrombin-fibrinogen complex does not provide information on the
contacts made by the natural substrate with exosite I. In the case of the interaction of thrombin with the
Bβ chain of fibrinogen, responsible for the release of FpB and protofibril polymerization, no structural
information is currently available.
This study fills an important structure-function gap in our understanding of the thrombin-
fibrinogen interaction. Here we report 3D-models of thrombin complexed with large fragments of the Aα
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and Bβ chains of fibrinogen, following a strategy recently used in the case of thrombin interaction with
the G-protein coupled receptors PAR1, PAR3 and PAR4 (3). The models are highly consistent with
available data on site-directed and naturally occurring mutants of thrombin and fibrinogen.
Materials and Methods
The N-terminal fragments of the fibrinogen Aα(20-62) and Bβ(31-72) chains were built, capped and
completed for missing hydrogens (pH 7.0) with Biopolymer and InsightII (Accelerys, San Diego CA) using
the human sequences P02671, P02675 and P02679 from the Swissprot sequence database (12). First,
500 models were produced with Modeller4 (13) by comparative modeling of every substrate on the
surface of thrombin using several available crystal structures of peptide-thrombin complexes as
templates. Substrate conformation was then optimized from the best model by simulated annealing
during molecular dynamic processes with Discover (Accelerys, San Diego, CA).
The fibrinogen fragment Aα(26-35) was threaded over the Ac-DFLAEGGGVR peptide from X-ray
crystal structures 1BBR and 1UCY (9,14). Residue R35 of the Aα chain was linked through its carbonyl O
to thrombin S195 Ογ to form a reaction intermediate analogous to the Arg of PPACK in 1PPB (15). The
conformation of fragment Aα(35-49) was templated on the protein fragments KILDKVVERIKERMKDSNVKL
from 1A0E and AFWRELVECFQKISKDSDCRA from 1DCI. These fragments were selected from our data-
base of 48 million conformers constructed by dissecting the entire Protein Data Bank into peptides from 3
to 18 residues in length. The conformers were screened for optimal shape, length and fit to the thrombin
surface. The conformation of fragment Aα(46-57) was loosely threaded over the NGDFEEIPEEYL hirugen
peptide from the structures 1HAH (16) and 1NRS (17). The fibrinogen fragment Bβ(41-44) was threaded
over the peptide LDPR from the structure 1NRS (17). Residue R44 of the Aα chain was linked through its
carbonyl O to thrombin Ser195 Oγ as described above. The conformation of fragment Bβ4(49-61) was
loosely threaded over the NGDFEEIPEEYL hirugen peptide from structures 1HAH and 1NRS and also over
the proline-rich peptide APTMPPPLPP from the structure 1ABO of a tyrosine kinase Src homology domain
3 (18).
Peptides were docked over thrombin using 1PPB and 1HAH as templates and yielded 500 models
that were ranked in terms of stereochemistry quality and lowest potential binding energies. Models
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containing a ligand with a r.m.s. deviation within 2 Å from a better ranked one were eliminated. We
selected the best ten models, extracted the ligand and docked it over thrombin in 1PPB as a starting
point. Water molecules and Na+ as defined in 1HAH and 1PPB were pooled and filtered for overlaps with
thrombin or ligand atoms and added to the starting model. The backbone of thrombin secondary
structures templated on 1PPB was kept frozen during simulated annealing applied by molecular dynamics
in cartesian space (fast annealing from 50 to 600 K in 3 ps, slow cooling from 600 to 50 K during 10 to
30 ps with progressive constraint to bring back thrombin backbone to the 1PPB reference). Models were
sampled every 1 ps. About 300 models (10 starts and 30 samples per trajectory) were minimized (200
steepest descent then 1500 conjugate gradient cycles). All processes were performed with the force field
CFF91 and Discover. Constraints were used to hold the Na+ coordination shell in place. The dielectric
constant was set at 1xr, cut offs were used to threshold non covalent bonds at 14 Å during Monte-Carlo
processes and dynamics, and set to infinite during minimizations.
Models of thrombin-substrate complexes with an r.m.s. deviation <1.5 Å for the thrombin
backbone and peptide residues <10 Å from the enzyme were considered acceptable. The stereochemistry
of final conformations was systematically checked with Procheck 3.0 (19) to satisfy a quality expected for
a 2.4 Å resolution model (the lowest resolution of the structures that we used as template). The solvent
accessible surface of thrombin was calculated according to the Connolly algorithm and rendered as a solid
surface with InsightII using a 1.4 Å radius probe.
Thrombin mutations were modeled with Biopolymer. Dihedral angles were explored with the
Monte-Carlo/Metropolis method for the main chains of the wild-type residue, its flanking residues, and
the side chains of residues within 6Å from the site of mutation. Minimizations were run in two steps with
Discover using the force field CFF91 and keeping the constraints on the Na+ and its coordination shell. A
steepest descent algorithm was applied during 300 cycles, followed by a conjugate gradient algorithm up
to 1500 cycles.
Thrombin sequences were selected from the non-redundant sequence database (NCBI, Bethesda
MY) with the BLAST program (20) and aligned with Clustal X (21). Sequences of thrombin natural
substrates were aligned according to the best model threaded and docked on the thrombin surface as
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described above, using one substrate fragment per orthologous group of sequences. Other orthologous
sequences were aligned over the multi-aligned sequence core with Clustal X.
Results
Interaction with the Aα chain. The biggest fragment from fibrinogen crystallized with thrombin is the
FpA(11-19) from bovine equivalent to human FpA(27-35) (9). Aα(26-39) is the longest fragment from
human fibrinogen bound to thrombin studied by NMR (10). Hirugen peptides inhibit fibrinogen cleavage
by binding to exosite I (16). We used these peptides as templates to model Aα(43-54). The three
structures 1BBR, 1UCY and 1HAH helped build the core F27LAEGGGVRGPRVVERHQSACKDSDWPF54 of the
human Aα(20-62) fragment. Equivalent positions of the human Aα(20-62) residues are not visible in the
crystal structure of the bovine fibrinogen Aα chain (5,8). The first documented motif is a helix starting at
position 63 in the corresponding human chain numbering. The helix end is accessible to solvent and we
therefore assumed that the N-terminal fragment 1-62 folds in a way accessible to thrombin. The
fragment is probably responsible for most of the interactions between fibrinogen and thrombin, because
Aα(20-70) produced by the cyanogen bromide hydrolysis of fibrinogen is a thrombin substrate as good as
the entire fibrinogen (22,23). We extended the templated peptide core to Aα(20-62) and docked it on
thrombin. The peptide is mainly in extended conformation with two short loops 28-33 (loop 1) and 39-48
(loop 2).
The Aα chain fragment explores different portions of the thrombin surface (Figure 1). Residues
20-25 interact with exosite II, residues 26-38 fit into the active site, residues 39-51 bind to the groove
connecting the active site with exosite I, and residues 52-62 dock onto exosite I. The change in exposed
surface area upon formation of the complex between thrombin and Aα(20-62) is 3501 Å2 (25% of the
thrombin surface), comparable to 3304 Å2 in the thrombin-hirudin interface (24). Half of the contact area
maps outside the region from the aryl binding site to the S3’ site framing the portion of substrate
interacting with the active site. The relevant contacts of the Aα(20-62)-thrombin complex are reported in
Figure 2 and compared to those of the templates.
There is a site of phosphorylation in the human fibrinogen Aα chain at AαS22 that results in an
intermediate showing higher specificity for thrombin (25). NMR analysis of the 31P 1D spectra changes
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due to binding to thrombin of the synthetic phosphorylated peptide Aα(20-41) suggests contribution of
this group to binding (26). Transferred nuclear Overhauser effect experiments locate the contact of this
group in the vicinity of R175 of thrombin, in exosite II. The phosphate seems to stabilize the otherwise
disordered residues Aα(22-26) in their interaction with thrombin (26). The backbone structure of residues
Aα(22-35) of our model of the Aα(20-62)-thrombin complex is superimposable to the bundle of NMR
conformers proposed by Maurer and colleagues (26). Our 3D-model suggests the interaction of the
phosphate group within the pocket formed by the basic side chains of R165, K169 and R175. AαD21
interacts with K169, and AαD26 with R97 to form ion-pairs. The naturally occurring mutant fibrinogen
Lille I (27) carries the substitution AαD26N and shows reduced clotting activity. The mutation should lead
to breakage of this ion-pair.
AαF27 is framed by W215, I174 and L99 forming the aryl binding site (15) and accounts for a
major fraction of the binding specificity of fibrinogen toward thrombin. The AαF27Y mutant abrogates
cleavage by thrombin in model peptides (28). A 3D-model of thrombin complexed with the Aα chain
carrying the F27Y mutation shows that the hydroxyl group of AαY27 H-bonds to the N atom of AαV34,
fixing the aromatic ring of AαY27 into a distorted position that pulls thrombin W215 1.4 Å higher and out
of the aryl binding site (Figure 3). This perturbation increases the solvent exposure of the hydrophobic
surface of the entire aryl binding site. Similar perturbations of W215 have been shown to produce drastic
(up to 500-fold) reduction in clotting activity (29). Furthermore, the steric hindrance introduced in the
thrombin mutant L99Y skews the AαF27 side chain away from the W215 ring, explaining the 260-fold
decrease in specificity for FpA release (3).
AαV34 is in close contact with thrombin Y60a and W60d in the S2/S3 sites. AαE30 is ion-paired
with R173, and the naturally occurring fibrinogen Mitaka II (30) shows reduced clotting activity
presumably because of the loss of this ion-pair.
AαR35 is the P1 residue of fibrinogen and is engaged in the expected ion-pair with D189 in the
S1 pocket via its side chain and a H-bond interaction with the carbonyl O of S214 through its N atom
(8,9). Mutation of this Arg to His as in fibrinogen Petoskey I (31), or Cys as in fibrinogen Metz I (32),
results in drastic or complete loss of clotting activity.
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Residues G31GG33 are important for positioning AαF27 and AαR35 in their respective pockets. The
mutation AαG31V in fibrinogen Rouen (33) causes reduced clotting activity. The intervening Gly in this
sequence makes a contact with the side chain of E217 through its carbonyl O atom. Removal of this
contact in the E217A mutant causes a significant loss of fibrinogen clotting (34). Removal of both this
contact and the strong coupling with W215 in the aryl binding site in the double mutant W215A/E217A
generates a thrombin mutant that has lost fibrinogen clotting, but retains activity toward the
anticoagulant protein C (35). The N atom of AαG33 H-bonds to the carbonyl O of G216.
Residues AαL28 and AαP37 frame W60d. The AαP37L substitution in fibrinogen Kyoto II (36) has
no effect on clotting. The model suggests that the Leu substitution can be accommodated well in the
hydrophobic environment between L41 and W60d of thrombin. On the other hand, the W60dS mutant of
thrombin shows significant perturbation of fibrinogen clotting (3,37), consistent with a drastic disruption
of the favorable hydrophobic interactions with AαL28 and AαP37 caused by the replacement of W60d
with a polar side chain.
Residue AαR38 makes a solvent accessible ion-pair with E39. The energetic contribution of this
ion-pair is modest because the E39A mutant of thrombin has clotting activity comparable to wild-type (3),
and the fibrinogen mutants AαR38S Detroit (38), AαR38G Aarhus (39) and AαR38N Munich (40) have
normal or slightly reduced clotting activity.
The Aα and Bβ chains have a loop segment between the anchor positioned into the S1 pocket
and the region binding to exosite I. Within this loop, AαC47 is exposed to solvent in the Aα(20-62)-
thrombin complex and is available for a disulfide bridge with AαC47 from the second Aα chain of the
fibrinogen dimer.
AαD51 is ion-paired with R67 in exosite I. The mutation R67A affects equally FpA and FpB
release (3), although there is no residue in the Bβ chain available for an ion-pair with R67. That suggests
that the loss of thrombin activity toward the Aα and Bβ chains is due to perturbation of the buried ion-
pair quartet involving R67, K70, E77 and E80 which holds the 70-loop and the all important residue Y76
in their correct conformation (15). Indeed the mutants R67A, K70A, E77A and E80A alter the kinetics of
FpA and FpB release drastically and to the same extent (3), even though K70 and E80 do not contact the
Aα or Bβ chains. Furthermore, the effect of these mutations is practically identical to that caused by the
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Y76A mutation (3), suggesting that the role of the buried quartet is to orient Y76 on the surface of the
70-loop for optimal interaction with fibrinogen.
The sequence W52PF54 of the Aα chain frames residue Y76 making a hydrophobic cluster with L65
and I82. AαP53 kinks the Aα chain into exosite I and enables optimal clipping of Y76 by the flanking
aromatic residues.
Other contacts made by residues of the Aα chain in this region are energetically dispensable. The
sequence D57ED59 of the Aα chain, also present in PAR1 as D58EE60 (3), makes ion-pairs with K81 and
K110 through AαD57, and with R77a through AαE58. However, the K81A, K110A and R77aA mutations
have little effect on the release of fibrinopeptides (3). Finally, the side chains of AαC55 and AαC65 are
accessible to solvent and available for disulfide bridging.
Interaction with the Bβ chain. The peptide Bβ(31-148) produced by cyanogen bromide hydrolysis is
not as good a substrate as the entire Bβ chain (41). We templated the conformation of F41SAR44 of the Bβ
chain by homology with the peptide LDPR complexed with thrombin in 1NRS (17). The Bβ chain also
shows a highly conserved proline-rich region in the range of contact with thrombin. The structures of
LDPR-hirugen-thrombin 1NRS (17), hirugen-thrombin 1HAH (16) and APTMPPPLPP bound to the SH3
domain 1ABO (18) were used to build the core F41SARGHRPLDKKREEAPSLRPAPPP65 of the human Bβ
chain fragment. We extended the peptide over its two ends to make a 3D-model of the fragment Bβ(31-
72) bound to thrombin. This portion of the Bβ chain is not visible in the available crystal structures (5,7).
The first motif visible is a helix starting at position 77 in the corresponding human chain numbering.
As for the Aα chain, the Bβ chain makes contact with an extended surface of thrombin (Figure
1). Residues 31-39 contact exosite II, residues 40-47 bind to the active site, residues 47-55 dock into the
groove connecting the active site to exosite I, and residues 56-72 interact with exosite I. The change in
exposed surface area when fragment Bβ(31-72) binds to thrombin is 3901 Å2 (28% of the thrombin
surface), a value that exceeds that of the Aα(20-62)-thrombin complex by 11%. Up to 60% of the
contact surface is located outside of the subsites from the aryl binding site to S3’. The relevant contacts
for the Bβ(31-72)-thrombin complex are reported in Figure 2 and compared to those of the templates.
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The terminal amine and BβG32 amide hydrogen interact with the carboxyl O atoms of D178. The
carbonyl O atoms of BβQ31 interact with the guanidinium group of R175. BβD35 interacts with K169,
BβE37 superimposes with AαE30 and makes a water-accessible ion-pair with R173, whereas BβE38 forms
a solvent accessible ion-pair with R97. As for the Aα chain, these interactions involve solvent exposed
residues and are expected to be energetically dispensable.
In the active site region, BβF41 superimposes well with AαF27 and fits in the aryl binding site
formed by W215, I174 and L99. Mutations of W215 or L99 have a drastic impact on the release of FpB
(3), underscoring the importance of this favorable hydrophobic interaction.
The side chain of BβF40 superimposes with AαL28 and is in close contact with Y60a and W60d in
the hydrophobic cluster acting as a lid over the S1 pocket. Mutation of W60d greatly affects the release
of FpB (3,37).
BβR44 is embedded into the S1 pocket, with its side chain ion-paired with D189 and its N atom
H-bonding to the carbonyl O atom of S214. Mutation of this Arg to Cys as in fibrinogen Ijmuiden (42)
abrogates FpB release, as expected.
The Gly residue in P1’ is conserved in all Aα and Bβ chains and comes into contact with G193.
The P1’ Gly is replaced by Cys in fibrinogen Ise (36), leading to a reduction in clotting activity. The BβH46
heterocycle at P2’ is staked against W60d, as are residues F43 in PAR1, F40 in PAR3 and Y49 in PAR4
(3). Residue BβR47 at the P3’ position makes a solvent exposed ion-pair with E39, whose energetic
contribution to recognition is dispensable in view of the properties of the E39A mutant (3).
Unlike the Aα chain, the Bβ chain does not possess an exposed Cys available for disulfide
bridging in the sequence upstream to the fragment that makes contact with exosite I. The carbonyl O
atom of BβP60 interacts weakly with the N atom of R67 in exosite I. As for the Aα chain, the loss of the
buried quartet involving R67, K70, E77 and E80 is responsible for the loss of specificity toward the Bβ
chain in the R67A, K70A or E80A mutations (3). The Bβ chain lacks aromatic groups around BβP63, unlike
the Aα chain. BβP58 and BβP61 frame F34 and BβP63, BβP64 and BβP65 cage Y76, in a face-to-face or
face-to-edge orientation.
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BβL59 points into the hydrophobic cluster formed by L65, Y76 and I82. The crucial involvement
of Y76 in all these interactions explains why the mutation Y76A has a drastic effect on the release of FpB,
as seen for the release of FpA (3).
Residues of the Bβ chain further downstream do not make relevant contacts with the thrombin
surface, except for the carbonyl O atom of BβI66 and the hydroxyl of BβS67 that H-bond to the charged
amine of K81. The sequence I66SGGG70 is absolutely conserved in all Bβ chains among different species.
Of these residues, BβY71 clusters with F114 and BβR72 ion-pairs with D116.
Discussion
The 3D-models of thrombin bound to fragments of the human fibrinogen Aα and Bβ chains are consistent
with available data on site-directed mutagenesis of thrombin, and with the phenotype observed with
several naturally occurring mutants of fibrinogen. These models also make it possible to align the
sequences of fibrinogens from other species and other thrombin natural substrates around their cleavage
site. The alignment (Figure 4) suggests a pattern of recognition by thrombin that applies generally to
other natural substrates. The two loops 1 and 2 fix the docking into the active site and groove region. In
the case of the fibrinogen Aα chain, loop 2 also provides room for the disulfide bridge with the
homologous Aα chain via AαC47. Acidic residues are frequently found in the region of the unprimed sites
that binds to exosite II. A hydrophobic/aromatic residue fills systematically the aryl binding site, as
documented in the crystal structures of thrombin bound to FpA (8,9) or PAR1 (17). A hydrophobic side
chain often precedes the residue bound to the aryl binding site (see for example the Aα and Bβ chains of
fibrinogen). The added hydrophobicity from this side chain provides optimal packing against the aromatic
moiety of W60d. In the primed sites, the P2’ position is often occupied by F, Y, H or P (noted ϕ in Figure
3) to frame the ring of W60d in sandwich with a Pro often found in the P2 position. Furthermore, a Pro
residue is often located downstream to the site of cleavage in a position to contact exosite I and appears
to be highly conserved among all thrombin substrates, with the exception of factor V, factor XIII and
TAFI. The role of this Pro (residue 53 in the case of the Aα chain) is to kink the substrate backbone into
exosite I at the thrombin surface. Hydrophobic/aromatic residues often flank this Pro (e.g., Aα chain,
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PAR3 and factor XI) and the Aα(20-60)-thrombin model indicates that the role of this aromatic-Pro-
aromatic triplet is to cage Y76 for optimal bridge-binding of exosite I and the active site.
There are 838 examples of aromatic-Pro-aromatic triplets in the Protein Database, of which 20%
show the aromatic residues pointing in the same direction and caging another aromatic side chain in 5%
of the cases. Interestingly, a screening of all sequences in the S1 family of serine proteases shows the
presence of a Pro residue located 13 residues downstream to the activation cleavage site in 83% of the
cases (the Pro is at position 27 in the standard chymotrypsin numbering). The origin of this conservation
is unclear, although the analogy with thrombin substrates discussed above should draw attention to a
possible “universal” role of this residue in optimizing the alignment of unprimed residues of substrate for
cleavage by the target protease. Site-directed mutagenesis of this conserved Pro in different systems will
be necessary to test this hypothesis.
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Figure legends
Figure 1. 3D-models of thrombin bound to fragments of the fibrinogen Aα (A,B) and Bβ (C,D) chains.
Thrombin is displayed as its solvent-accessible surface based on the coordinates 1PPB (15). The
fibrinogen fragments Aα(20-62) and Bβ(31-72) are rendered as solid sticks in purple. The orientation is
with the thrombin active site at the center (A,C), or exosite I at the center (B,D). The thrombin surface is
color-coded to reflect the extent of perturbation induced by the mutation of specific residues (labeled in
the top panel) on the value of the specificity constant kcat/Km for the release of FpA or FpB (3). The scale
is: red (-3.5÷-2.5 log units), orange (-2.5÷-1.5 log units), green (-1.5÷-0.5 log units), cyan (-0.5÷0.5 log
units), blue (0.5÷1.5 log units). Residue K70 is buried under Y76 in exosite I and is not visible.
Figure 2. Bound fragments Aα(20-57) (blue in A and B) and Bβ(31-68) (magenta in C and D) displayed
over the thrombin surface of the active site (A and C) and exosite I (B and D) and superimposed to the
peptides used as templates: DFLAEGGGVR from 1BBR (green in A), hirugen NGDFEELPEEYL from 1HAH
(green in B and D), LDPR from 1NRS (green in C). The r.m.s. deviations between the backbones of
fragments Aα and Bβ relative to hirugen are 1.44 Å and 0.41 Å, respectively. Contacts with thrombin
residues <6 Å are listed in E for Aα(20-57), Bβ(31-68), and the templates LDPR and hirugen. Sequences
in E are aligned according to the 3D-models of complexes. The position of phosphate groups and
disulfide bridges are indicated.
Figure 3. 3D-models of the fibrinogen fragments (with residues numbered in blue) Aα(20-57) for wild-
type (blue) and the F27Y mutant (green), displayed over the thrombin surface of the active site (with
residues numbered in black). The F27Y mutation introduces a H-bond between the hydroxyl group of Y27
and the N atom of V34, which fixes the aromatic ring of Y27 into a distorted position that pulls thrombin
W215 1.4 Å higher and out of the aryl binding site. This perturbation results in a non-favorable increase
in the solvent exposure of the hydrophobic surface of the entire aryl binding site around W215.
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Figure 4. Thrombin substrate sequences docked over thrombin and aligned from their 3D-superposition.
Sequences refer to human unless otherwise specified. Orthologous Aα and Bβ chains of fibrinogen were
aligned below human sequences with Clustal X. The position of the first residue is indicated in the second
column. Sequence and structure files in the last column are specified from SwissProt, Genbank and
Protein Data Bank.
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C
A
Figure 1 Rose & Di Cera, 2002
D
B
G149d
R77a
Y76
R75
R67
W148
R73
E192E217
A149a
V149c
K149e
T147
W60d
W96
W215
R221a
K224
L99
Y225
M84
K110
I82
Y76R77a
I24R75
R67
R73
K149e
L65
W60d
E192
T147
W148
V149c
A149a
Y117
E217
R35
E39
K36
S36a
Q38F34
R35 S36a
K36
Q38
K81
F34
T74T74
W96
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W60d
W60d
F34
F34
L99
L99
I174
R173
R173 R67Y76
Y76
Figure 2 Rose & Di Cera, 2002
C
A
D
B
W215
W215
Y60a
Y60a
Q38
Q38
E192
E192
L65
M84
M84
L65I82
I82
F41 F40
R44
P63
P64
L1
D2
P3
E37
F56
I59
P60
Y63L64
K36
W52
P53 F54
Y63L64
I59
F56
D49
E57
R73
E57
R73
C47C55E30
L28
D26
F27
R35
G31 G32
V34
E38
G39
H46
R38 exo
site
IIac
tive
sit
eS
’ gro
ove
exo
site
I
Q31 D178G32V33 I176
A20 N34D21 K169 D35 K169S22(PO4
- ) R165 K169 R175 N36 G23 E37 R173E24 R175 E38 R97G25 G39D26 R97 F40 Y60a W60dF27 L99 I174 W215 F41 L99 I174 W215 L1 L99 I174 W215L28 Y60a W60d - -A29 - -E30 R173 - -G31 - -G32 E217 - -G33 G216 S42 E192 D2V34 Y60a W60d A43 Y60a P3 Y60a W60dR35 H57 D189 S195 S214 R44 H57 D189 S195 S214 R4 H57 D189 S195 S214G36 G193 G45 G193P37 H57 W60d H46 W60dR38 D60e R47 D60eV39 L40 P48V40 L41 L49 L41E41 D50- K51 D60eR42 E192 K52H43 F34 L41 R53 E35 L40Q44 E54S45 K73 E55A46 A56 N53C47-(C47α) P57 Q51 G54K48 &74 S58 D55- L59 -D49 R67 R60 F34 F56 F34S50 P61 E57 R73D51 A62 E58W52 L65 Y76 P63 L65 Y76 I59 L65 Y76P53 Y76 P64 Y76 P60 Y76F54 Y76 I82 P65 Y76 I82 E61C55-(C95β’) I66 I118 E62S56 K81 S67 K81 Y63 Y76 I82D57 G68 L64 L65 I82 K36
Aα thrombin Bβ thrombin E
ldpr thrombin
hir thrombin
R4
D60e
K51
D60e
V40
V39
K36
I118
K81
K81
D55
E58
P61
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L99
R173
Figure 3 Rose & Di Cera, 2002
W215
Y60a
E192
E30
L28
D26
F27
R35G31
G32
V34
Y27
W60d
G25
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Aα 20 ADSGEG DFLAEGGGVR GPR VVE---RHQSACK -DSDWPFCSD EDWNYKCPS P02671Aα bovine 1 EDGSDPPSG DFLTEGGGVR GPR LVE---RQQSACK -ETGWPFCSD EDWNTKCPS P02672Aα chicken 1 AQDGKT TFEKEGGGGR GPR ILEN--MHESSCK YEKNWPICVD DDWGTKCPS P14448Aα rat 3 ATGTTS EFIEAGGDIR GPR IVE---RQPSQCK -ETDWPFCSD EDWNHKCPS P06399
Bβ 31 QGVNDNEEG FF-----SAR GHR PLD--KKREEAPS LRPAPPPISG GGYRARPAK P02675Bβ bovine 10 QDDRPKV GL-----GAR GHR PYD--KKKEEAPS LRPVPPPISG GGYRARPAT P02676Bβ rat 8 ATATDSDKV DL----SIAR GHR PVD--RRKEEPPS LRPAPPPISG GGYRARPAK P14480
PAR1 26 ARRPASKATNA TL-----DPR SFL LR--------NPN DKYE-PFWED EEKNESGLT P56488PAR3 23 MENDTNNLAKP TL-----PIK TFR GA--------PPN SFEEFPFSAL EGWTGATIT O00254PAR4 39 STGGGDDSTPS IL----PAPR GYP GQ-----VCANDS DTLELPDSSR ALLLGWVPT AAC28860fII 184 PVCGQD-QVTV AM-----TPR SEG SS-----VNLSPP LEQCVPDRGQ QYQGRLAVT P00734fII 312 EGRTATSEYQT FF-----NPR TFG SG---------EA DCGLRPLFEK KSLEDKTER P00734fV 722 ADYDYQNRLAA AL-----GIR SFR NSS-------LNQ EEEEFNLTAL ALENGTEFV P12259fV 1031 KKKKEKHTHHA PL-----SPR TFH PLRS-EAYNTFSE RRLKHSLVLH KSNETSLPT P12259fVII 197 KIPILEKRNAS KP-----QGR IVG GK---------VC PKGECPWQVL LLVNGAQLC P08709fVIII 376 VVRFDDDNSPS FI-----QIR SVA KKHPKTWVHYIAA EEEDWDYAPL VLAPDDRSY P00451 fVIII 744 DISAYLLSKNN AI-----EPR SFS QNSRHPSTRQKQF NATTIPENDI EKTDPWFAH P00451fXI 372 RLCKMDNECTT KI-----KPR IVG GT---------AS VRGEWPWQVT LHTTSPTQR P03951fXIII 18 NNSNAAEDDLP TVELQGVVPR GVN LQ-------EFLN VTSVHLFKER WDTNKVDHH Q9NQP5PC 199 RDKRDTEDQED QV-----DPR LID GK---------MT RRGDSPWQVV LLDSKKKLA P04070TAFI 99 EDLIQQQISND TV-----SPR ASA SYYE-----QYHS LNEIYSWIEF ITERHPDML g4503005
Consensus -- L ins1 PR Gφ+ ins2 -- φPφ
Thrombin contacts
Y76
R67
D18
9
L65 I82
I174
W21
5L
99
K16
9R
173
D60
eE
39
Figure 4 Rose & Di Cera, 2002
W60
d
exosite II active site S’ groove exosite I
Y60
a W
60d
G19
3
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Thierry Rose and Enrico Di Cera3D-modeling of thrombin-fibrinogen interaction
published online March 18, 2002J. Biol. Chem.
10.1074/jbc.M110977200Access the most updated version of this article at doi:
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