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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: enrico@biochem.wustl.edu

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.

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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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