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Molecular Basis of Thrombin Recognition by Protein C Inhibitor Revealed by the 1.6-Å Structure of the Heparin-Bridged Complex

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Molecular Basis of Thrombin Recognition by Protein C Inhibitor Revealed by the 1.6-Å Structure of the Heparin-Bridged Complex Molecular basis of thrombin recognition by protein C inhibitor revealed by the 1.6-Å structure of the heparin-bridged complex Wei Li*, Ty E. Adams*, Jyoti Nangalia*, Charles T. Esmon†, and James A. Huntington*‡ *Department of Haematology, Division of Structural Medicine, Thrombosis Research Unit, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, United Kingdom; and †Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Departments of Pathology and Biochemistry and Molecular Biology, Howard Hughes Medical Institute, University of Oklahoma Health Science Center, Oklahoma City, OK 73104 Edited by Shaun R. Coughlin, University of California, San Francisco, CA, and approved February 4, 2008 (received for review November 21, 2007) Protein C inhibitor (PCI) is a serpin with many roles in biology, where the rate of inhibition depends on the formation of the including a dual role as pro- and anticoagulant in blood. The initial recognition (Michaelis) complex, and in the second step protease specificity and local function of PCI depend on its inter- both serpin and protease undergo significant conformational action with cofactors such as heparin-like glycosaminoglycans rearrangement leading to a covalently linked final complex. The (GAGs) and thrombomodulin (TM). Both cofactors significantly serpin mechanism is especially well suited for inhibiting coagu- increase the rate of thrombin inhibition, but GAGs serve to pro- lation factors because the conformational change induced in the mote the anticoagulant activity of PCI, and TM promotes its protease can result in a loss of cofactor binding. An important procoagulant function. To gain insight into how PCI recognition of example of this is the induced disorder of exosite I on thrombin thrombin is aided by these cofactors, we determined a crystallo- upon complexation with serpins (13, 14). Thrombin can thus be graphic structure of the Michaelis complex of PCI, thrombin, and removed from its high-affinity interaction with TM through heparin to 1.6 Å resolution. Thrombin interacts with PCI in an inhibition by PCI. Regulation of serpin activity by cofactors is unusual fashion that depends on the length of PCI’s reactive center generally achieved by altering the rate of formation and stability loop (RCL) to align the heparin-binding sites of the two proteins. of the Michaelis complex (15). Heparin-like GAGs accelerate BIOCHEMISTRY The principal exosite contact is engendered by movement of the rate of APC and thrombin inhibition by PCI, and TM thrombin’s 60-loop in response to the unique P2 Phe of PCI. This selectively accelerates the inhibition of thrombin. Heparin is mechanism of communication between the active site of thrombin thought to bridge PCI to thrombin in a manner similar to that and its recognition exosite is previously uncharacterized and may recently seen in the crystal structure of the antithrombin (AT)- relate to other thrombin substrate–cofactor interactions. The co- thrombin-heparin ternary complex (16, 17), but the APC-PCI factor activity of heparin thus depends on the formation of a bridged complex may involve a more intimate shared site on heparin-bridged Michaelis complex and substrate-induced exosite heparin (18, 19). In either case, cofactor activity depends on both contacts. We also investigated the cofactor effect of TM, estab- serpin and protease binding to the same heparin chain, with lishing that TM bridges PCI to thrombin through additional direct acceleration of inhibition conferred by both improved rate of interactions. A model of the PCI–thrombin–TM complex was built and diffusion and stabilization of the encounter complex by the evaluated by mutagenesis and suggests distinct binding sites for heparin bridge. The cofactor activity of TM relies on its tight heparin and TM on PCI. These data significantly improve our under- interaction with thrombin and possible exosite interactions standing of the cofactor-dependent roles of PCI in hemostasis. between TM and PCI. Thrombin is known to bind to the EGF domains 5 and 6 of TM, and PCI is thought to interact with EGF hemostasis ͉ serpin ͉ crystallography ͉ thrombomodulin ͉ exosite domain 4 (20), although a direct interaction between TM and PCI has not been demonstrated. rotein C inhibitor (PCI) is a member of the serpin family of The structures of native (18) and reactive center loop (RCL)- Pprotease inhibitors (for recent reviews see refs. 1 and 2), first cleaved (19) PCI revealed the general serpin fold and some described for its ability to inhibit the anticoagulant-activated important additional features relevant to cofactor and protease protein C (APC) (3, 4). The procoagulant function of plasma binding. The region recognized by proteases as a substrate loop, PCI was further supported by the finding that the thrombin– the RCL, is unusually long and flexible in PCI. This can thrombomodulin (TM) complex, responsible for activation of influence protease binding in two important ways: First, the rate protein C, is also selectively inhibited by PCI (5). However, PCI of Michaelis complex formation will be reduced because of the is also capable of inhibiting several of the procoagulant proteases inherent mobility of the RCL. In addition, there will be a large generated as part of the hemostatic response, raising the possi- entropic cost for RCL binding by a protease, predictably reduc- bility of a dual role for PCI in regulating blood coagulation (for ing the stability of the Michaelis complex. These factors combine K for proteases, a fact borne out by the generally reviews see refs. 6 and 7). Elevated PCI levels have been to increase the m associated with venous thrombosis (8) and myocardial infarction (9), and the PCI–APC complex level is a sensitive marker for Author contributions: W.L. and J.A.H. designed research; W.L. and J.N. performed research; thrombotic events (10). The function of PCI in hemostasis T.E.A. and C.T.E. contributed new reagents/analytic tools; W.L. and J.A.H. analyzed data; depends on cofactors (for review see ref. 11). Heparin and and J.A.H. wrote the paper. similar glycosaminoglycans (GAGs) accelerate the inhibition of The authors declare no conflict of interest. several proteases, including APC and thrombin, and TM accel- This article is a PNAS Direct Submission. erates the inhibition of thrombin. Determining how these co- Data deposition: Coordinates and structure factors are deposited in the Protein Data Bank, factors influence the rate of protease inhibition by PCI is critical www.pdb.org (PDB ID code 3B9F). to understanding the various roles of PCI in regulating blood ‡To whom correspondence should be addressed. E-mail: [email protected]. coagulation. This article contains supporting information online at www.pnas.org/cgi/content/full/ PCI utilizes the well characterized serpin mechanism of pro- 0711055105/DCSupplemental. tease inhibition (12). It is best described as a two-step reaction © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711055105 PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 ͉ 4661–4666 Downloaded by guest on September 26, 2021 low rates of protease inhibition by PCI, and necessitate cofactors Table 1. Data processing, refinement, and model (3B9F) and/or exosite interactions to improve the Km. The second function of a long, flexible RCL is to increase the reach of a Crystal tethered protease to engage exosites on a larger surface of PCI. Space group P1 Cell dimensions, Å a ϭ 44.05; b ϭ 48.82; c ϭ 97.87 Another structural difference between PCI and other serpins is ␣ ϭ ␤ ϭ ␥ ϭ the location of the heparin-binding site along helix H (18, 21–23). Angles, ° 78.73; 81.49; 77.68 All other heparin-binding serpins (e.g., antithrombin, heparin Solvent content, % 43.9 cofactor II, plasminogen activator inhibitor I, protease nexin 1) Data processing statistics use a basic helix D for heparin binding (11). The heparin-binding Wavelength, Å 1.06 (Diamond, beam line I03) site of PCI on helix H is near to the RCL and thus in close Resolution, Å 37.50–1.60; 1.69–1.60 proximity to an attacking protease. This can have additional Total reflections 305,107; 25,620 effects on protease recognition, such as abrogating (24) or Unique reflections 95,914; 13,603 Multiplicity 3.2; 1.9 accelerating complex formation by altering the surface of PCI ͗ ␴ ͘ encountered by proteases. I/ (I) 12.9; 1.8 To understand how PCI recognizes thrombin and the accel- Completeness, % 93.6; 90.3 erating effect of cofactors, we determined a 1.6-Å crystal Rmerge* 0.052; 0.445 structure of the Michaelis complex between thrombin and PCI Model in the presence of a bridging heparin chain. Thrombin is docked Number of atoms modeled on PCI in an unusual position that aligns its heparin binding site Protein 5,493 (exosite II) with that of PCI (helix H). The position of thrombin Heparin 31 is allowed by the extra RCL residues C-terminal to the P1–P1Ј Water 523 bond [substrate residues are numbered Pn N-terminally and PnЈ Carbohydrate 24 C-terminally of the P1–P1Ј scissile bond, and the corresponding Glycerol molecules 5 Ј Sulphate Ions 3 cavities in the protease are numbered Sn and Sn (25)] and 2 engenders significant exosite interactions that help stabilize the Average B-factor, Å 30.2 complex. A model of the PCI–thrombin–TM Michaelis complex Refinement statistics 30.95–1.60 Å; 1.70–1.60 Å Reflections in working/free set 92,977/2,928; 15,020/460 was built on the structure and suggested favorable direct inter- 2 actions between TM and PCI that were supported by mutagen- R-factor /R-free, % 20.8/23.4; 32.5/33.7 esis and biochemical studies.
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