Three-dimensional Modeling of Thrombin-Fibrinogen Interaction

Rose, Thierry; Di, Enrico

doi:10.1074/jbc.m110977200

Cited by 56 publications

(48 citation statements)

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“…Hirudin blocks access to the active site of thrombin using its compact N-terminal domain and binds to exosite I via its extended acidic C-terminal domain. The mode of interaction of the C-terminal domain of hirudin was later documented in the structures of thrombin bound to hirugen (49), fibrinogen (8,9,50), PAR1 (12,13), PAR3 (15), and thrombomodulin (51). The structure presented here in the complex with the extracellular fragment of PAR1 expands our knowledge of how thrombin recognizes macromolecular substrates at both the active site and exosite I.…”

Section: Discussionmentioning

confidence: 75%

Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

Gandhi

Chen

2010

Journal of Biological Chemistry

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108

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Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here, we present the 1.8 Å resolution structure of thrombin S195A in complex with a 30-residue long uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. The structure reveals two unexpected features of the thrombin-PAR1 interaction. The acidic P3 residue of PAR1, Asp 39 , does not hinder binding to the active site and actually makes favorable interactions with Gly 219 of thrombin. The tethered ligand domain shows a considerable degree of disorder even when bound to thrombin. The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates.Thrombin is a trypsin-like protease endowed with important physiological functions that are mediated and regulated by interaction with numerous macromolecular substrates, receptors, and inhibitors (1). Fenton (2) was the first to recognize that thrombin selectivity toward macromolecular substrates depends on interactions with exosites that extend beyond the active site. Exosite I is positioned some 15 Å away from the active site (3) and occupies a domain analogous to the Ca 2ϩ -binding loop of trypsin and chymotrypsin (4). Recognition of macromolecular substrates or receptors responsible for the procoagulant, prothrombotic, signaling, and anticoagulant functions of thrombin depends on the structural integrity of this exosite (1). In general, exosite-dependent binding is kinetically limiting as a recognition strategy of macromolecular targets by enzymes involved in blood coagulation (5). Structural and site-directed mutagenesis data document the important role of exosite I in the interaction of thrombin with fibrinogen (6 -9) and the protease-activated receptors (PARs) 2 PAR1 (7, 10 -13) and PAR3 (7,14,15). Likewise, abundant structural and functional data document how thrombin recognizes substrates at the active site (3, 16, 17), including fibrinogen (18), PAR4 (15,19), and factor XIII (20, 21). On the other hand, structural elucidation of how substrates bridge the active site and exosite I in their binding to thrombin has been challenging. PAR1 is a premiere prothrombotic and signaling factor (22), the most specific physiological substrate of thrombin in terms of k cat /K m values (7), and a most relevant target for crystallization studies. PARs are members of the G-protein-coupled receptor superfamily and play important roles in blood coagulation, inflammation, cancer, and embryogenesis (23-28). Four PARs have been cloned, and they all share the same mechanism of ...

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Section: Discussionmentioning

confidence: 75%

Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

Gandhi

Chen

2010

Journal of Biological Chemistry

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108

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“…Although previous reports (17,26) had provided structural details on binding of FPA to the active site cleft of thrombin, the exact nature of the interaction between fibrin(ogen) and thrombin exosite I could only be surmised. This was partially clarified by molecular modeling of thrombin in complex with the NH 2 -terminal regions of the fibrinogen A␣ and B␤ chains (44). In this study, we solved the crystal structure of thrombin in complex with the fibrinogenderived thrombin-treated E ht fragment and established the mode of their interaction via thrombin exosite I.…”

Section: Discussionmentioning

confidence: 99%

“…binding, although a recent molecular modeling study suggests that it interacts with the SЈ groove of thrombin connecting the active site with exosite I (44). On the other hand, the B␤15-55 region is clearly involved because fibrin lacking B␤15-42 residues shows reduced thrombin binding (35,49).…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of the complex between thrombin and the central “E” region of fibrin

Pechik

Madrazo

Mosesson

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

114

104

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Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized D-Phe-ProArg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E ht, corresponding to the central region of human fibrin, and solved its structure at 3.65-Å resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E ht in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E ht. Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A␣ chain Trp-33, Phe-35, Asp-38, Glu-39, the B␤ chain Ala-68 and Asp-69, and the ␥ chain Asp-27 and Ser-30 of E ht form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.

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“…The compact N-terminal domain of the inhibitor occludes the active site of the enzyme and contacts the 60-loop, the aryl binding site, and the Na ϩ site (22, 23). These are the same regions probed by fibrinogen and PAR1 upon binding to thrombin (24,25). Last, but not least, hirudin binding to thrombin is positively linked to Na ϩ binding (19,26).…”

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confidence: 99%

Hirudin Binding Reveals Key Determinants of Thrombin Allostery

Mengwasser

Bush

Shih

et al. 2005

Journal of Biological Chemistry

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Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S3 F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na ؉ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na ؉ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.The serine protease thrombin is a member of the large class of enzymes activated by monovalent cations (1) and requires Na ϩ for optimal catalytic activity. The effect of Na ϩ is allosteric (2) and shifts the conformation of the enzyme from a low activity slow (S) form to a high activity fast (F) form. The partitioning of thrombin between these two forms is of physiologic importance because the Na ϩ -bound F form accounts for the procoagulant (cleavage of fibrinogen) and prothrombotic/ signaling (cleavage of PAR1 and platelet activation) roles of the enzyme, whereas the Na ϩ -free S form is responsible for the anticoagulant (cleavage of protein C) role (3, 4). Ancillary procoagulant roles of thrombin, like activation of factor VIII, also require the enzyme to be in the F form (5). The Na ϩ -dependent allosteric regulation of catalytic activity is shared by other clotting proteases, such as activated protein C (6, 7) and factor Xa (8), that possess a Na ϩ binding site structurally similar (7, 9) to that found in thrombin (10). Na ϩ binding to thrombin increases both the rate of substrate diffusion into the active site and the rate of substrate acylation (2, 11). The result is that the F form interacts with chromogenic substrates, fibrinogen, and PAR1 with significantly higher k cat /K m compared with the S form (4). Recent crystal structures of the S and F forms have helped rationalize this difference (12). Na ϩ binding causes rearrangements of residues close to the Na ϩ site and located up to 15 Å away such as the catalytic Ser-195. The long-range communication of the Na ϩ effect is ensured by a network of water molecules that embed the Na ϩ site, the primary specificity pocket, and the active site. Asp-189 at the bottom of the primary specificity pocket is o...

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Three-dimensional Modeling of Thrombin-Fibrinogen Interaction

Cited by 56 publications

References 41 publications

Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

Crystal structure of the complex between thrombin and the central “E” region of fibrin

Hirudin Binding Reveals Key Determinants of Thrombin Allostery

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