Crystal Structure of Thrombin in a Self-inhibited Conformation

Abstract

The activating effect of Na+ on thrombin is allosteric and depends on the conformational transition from a low activity Na+-free (slow) form to a high activity Na+-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na+ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na+ site and the active site regions. Here, we report a 1.87Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow → fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na+-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na+ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215. The activating effect of Na+ on thrombin is allosteric and depends on the conformational transition from a low activity Na+-free (slow) form to a high activity Na+-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na+ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na+ site and the active site regions. Here, we report a 1.87Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow → fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na+-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na+ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215. Thrombin is a Na+-activated enzyme (1Wells C.M. Di Cera E. Biochemistry. 1992; 31: 11721-11730Crossref PubMed Scopus (226) Google Scholar) that belongs to the vitamin K-dependent family of serine proteases involved in blood clotting (2Davie E.W. Kulman J.D. Semin. Thromb. Hemostasis. 2006; 32: 3-15Crossref PubMed Scopus (213) Google Scholar, 3Page M.J. MacGillivray R.T.A. Di Cera E. J. Thromb. Hemostasis. 2005; 3: 2401-2408Crossref PubMed Scopus (60) Google Scholar). As for other enzymes activated by monovalent cations (4Di Cera E. J. Biol. Chem. 2006; 281: 1305-1308Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), the binding of Na+ facilitates substrate binding and catalysis by lowering the energies of complex formation in the ground and transition states (5Ayala Y.M. Di Cera E. Protein Sci. 2000; 9: 1589-1593Crossref PubMed Scopus (46) Google Scholar, 6Krem M.M. Prasad S. Di Cera E. J. Biol. Chem. 2002; 277: 40260-40264Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The mechanism of Na+ activation is allosteric, or Type II (4Di Cera E. J. Biol. Chem. 2006; 281: 1305-1308Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and depends on the equilibrium between two active forms, slow (Na+-free) and fast (Na+-bound), that differ in their Michaelis-Menten parameters (1Wells C.M. Di Cera E. Biochemistry. 1992; 31: 11721-11730Crossref PubMed Scopus (226) Google Scholar). Na+ increases both kcat and kcat/Km and acts as a “modifier” (7Botts J. Morales M. Trans. Faraday Soc. 1953; 49: 696-707Crossref Scopus (125) Google Scholar, 8Page M.J. Di Cera E. Physiol. Rev. 2006; 86: 1049-1092Crossref PubMed Scopus (235) Google Scholar) that converts the low activity slow form into the high activity fast form. Crystal structures of the active slow and fast forms have been solved recently (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Significant changes have been detected between the two forms that explain how Na+ binding causes faster diffusion of substrate into the active site and faster acylation. A highly organized network of water molecules connects the coordination shell of Na+ to the hydroxyl group of the catalytic Ser-195 located 16 Å away, using the side chains of Asp-189 and Glu-192 as relays (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The correct orientation of Asp-189 in the primary specificity pocket and Ser-195 in the active site are controlled long range by Na+ binding and result into an optimal environment for substrate diffusion and catalysis. Earlier stopped-flow kinetic measurements of Na+ binding to thrombin (10Lai M.T. Di Cera E. Shafer J.A. J. Biol. Chem. 1997; 272: 30275-30282Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) have suggested that the mechanism of conversion of the slow to the fast form must involve a third component. The kinetics of Na+ association with thrombin was found to be biphasic, with a “fast” phase caused by the interaction of Na+ with the slow form S to generate the fast form F and a “slow” phase caused by the interconversion between the inactive S* and active S slow forms. The inactive S* form was found incapable of binding Na+ and substrate at the active site (10Lai M.T. Di Cera E. Shafer J.A. J. Biol. Chem. 1997; 272: 30275-30282Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Following the pioneering structure of thrombin free of inhibitors and Na+ portraying the active slow form (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 11Pineda A.O. Savvides S.N. Waksman G. Di Cera E. J. Biol. Chem. 2002; 277: 40177-40180Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), a number of crystal structures of free thrombin have been obtained with the enzyme in various inactive forms (12Huntington J.A. Esmon C.T. Structure (Camb.). 2003; 11: 469-479Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13Carter W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, A.O. Chen Z.W. Caccia S. Savvides S.N. Waksman G. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar). of these structures W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar) have been to the of the inactive slow form with the active site and the Na+ site structure has suggested that the of the inactive slow form of thrombin be to the Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) for interaction with 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of an inactive slow form is and to thrombin of the structures portraying thrombin into inactive conformations in the absence of Na+ that two molecules in the asymmetric unit in packing interactions at the active site and the Na+ site regions that into question the to the of The of the inactive slow form of thrombin to be with structures where packing interactions the conformation of the enzyme the Na+ site and the active site. Here, we report a high resolution crystal structure of in the absence of inhibitors and with the enzyme in an unprecedented self-inhibited conformation that is the result of crystal packing a the → of the catalytic to thrombin with the of the catalytic and to a for in the absence of inhibitors or in complex with was by the crystal structure of the in the presence of S. T. J. W.J. C.S. PubMed Scopus Google Scholar), is to that of the shows a in catalytic The thrombin was and for activity as (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Y.M. Di Cera E. Sci. S. PubMed Scopus (47) Google Scholar). The kcat/Km for the hydrolysis of the substrate is to at of a in kcat measurements of Na+ binding to thrombin and with an using an of and of of thrombin at a of with of the at of to at a of with The was with in the The Na+ binding has an phase that be the of the by a single slow phase with a that as increases The mechanism of Na+ binding the kinetic (10Lai M.T. Di Cera E. Shafer J.A. J. Biol. Chem. 1997; 272: 30275-30282Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google of Na+ binding to thrombin and in the presence of both the mechanism of binding is biphasic, with a fast phase by a slow phase of with or The to the effect of the for the slow phase of by stopped-flow measurements of Na+ binding to thrombin and The of with that the slow phase Na+ binding and to the conformational transition between S and S* kinetic and The to in the with The of slow forms in the inactive is for and for the The phase is to Na+ binding to S to generate with an equilibrium association The slow phase is to the conversion of S* to S with an (10Lai M.T. Di Cera E. Shafer J.A. J. Biol. Chem. 1997; 272: 30275-30282Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), where and are the for the S* → S transition and and the of inactive slow form to the active slow form as The thrombin W215P was and for activity as (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Y.M. Di Cera E. Sci. S. PubMed Scopus (47) Google Scholar). of and and activation of protein C as (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Y.M. Di Cera E. Biochemistry. 2000; PubMed Scopus (47) Google Scholar, Di Cera E. Biochemistry. PubMed Scopus Google Scholar, Bush L.A. A.O. Caccia S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) of at the was to in and was using the diffusion by of protein and of and to at The with group and unit parameters a Å and and molecule in the asymmetric The crystal was in and to to Å resolution at on an at the of the and was using the 1997; PubMed Scopus Google Scholar). The was at the Å resolution with at the resolution The of are in of the thrombin range from and and in of from and and in of side protein using from and and in of side using 1992; PubMed Scopus Google Scholar). in a The crystal structure of thrombin was solved by using the of the slow form of thrombin to (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), as the The structure was using a and J. M. T. Biol. PubMed Scopus Google Scholar), and of the as a for of was at Å by slow and A of and and at Å The resolution for was to and water molecules at the of using The and and with from of Å for and for The and water The be of and was The and parameters are in The for thrombin shows no in the with of the located in the regions and located in the regions. The of the structure of the thrombin have been in the Protein of the structures portraying thrombin in inactive conformations in the absence of inhibitors and Na+ W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, A.O. Chen Z.W. Caccia S. Savvides S.N. Waksman G. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar) two molecules in the asymmetric by that The packing the active site and the Na+ site and into question the to the absence of found at the of these structures are in and the involved in the packing are in Å of thrombin structures in the Na+-free number of molecules in the asymmetric at the The number of molecules in the asymmetric in a The recently solved structure of free thrombin in an inactive conformation to the of Na+ Huntington J.A. J. 2005; PubMed Scopus Google Scholar) has two molecules in the asymmetric unit an of interaction of The of the and and is the of the of the complex between thrombin and the of Y.M. M. M.T. Di Cera E. J. Biol. PubMed Scopus (47) Google Scholar). is a structure of thrombin by thrombin molecule in the asymmetric with the Na+ site and active site regions the of significant crystal was to a Å in the of that to the active site Huntington J.A. J. 2005; PubMed Scopus Google Scholar). Glu-192 is at the of the two thrombin molecules in the asymmetric with the Å from the of Arg-221a of the The the side of Glu-192 into the active where is further by two with a molecule at the The conformation of Glu-192 may have a of the of the with the of the catalytic the be a of the inactive slow form is also in the structure of thrombin to C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar). significant packing interactions the involve with with and with to be involved in the allosteric transition from the inactive slow form to the active fast form Huntington J.A. J. 2005; PubMed Scopus Google Scholar). of a with of and the catalytic forms a with a molecule at the the inactive conformation in structure is the result of crystal to the structure of the thrombin W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar), also to thrombin in the inactive slow form. packing interactions are and a Å in the Na+ site and active site regions The Å between of Glu-192 and of Arg-221a of the molecule in the asymmetric unit Glu-192 the active site. of the residue forms a Å with of in the molecule of the asymmetric to the that side a significant in the structure W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The inactive conformation in structure is the result of the the Na+ site and significant crystal The structure of the thrombin in the presence of two molecules in the asymmetric with to the Na+ site and the other in an inactive conformation with the Na+ site free Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar). structure packing interactions in the asymmetric unit that a Å The of molecule is at the and its the the of molecule is by its with molecule at the is in with Arg-221a of molecule explain of the the and of the to the primary specificity of have also caused the of into the primary specificity pocket, as in the structure of the inactive A.O. Chen Z.W. Caccia S. Savvides S.N. Waksman G. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), as as the in the side of Trp-215 that is also in the structure of inactive thrombin Huntington J.A. J. 2005; PubMed Scopus Google Scholar). As in the of other structures to thrombin in the inactive slow form W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar), the inactive conformation of molecule in the structure in the presence of C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar) is the result of crystal may be that crystal are the the of changes C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar), be highly to have structures of the inactive slow form where packing interactions involve regions by the allosteric slow → fast transition (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The structure of the thrombin a significant in The structure a single molecule in the asymmetric with with molecules in the The of is to with a of Å with the Na+-bound fast form (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). are no significant changes at the site of mutation that be to the of of and of as for (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). the other the structure shows changes in the primary specificity pocket and in the Na+-binding site The into the primary specificity is Å from the of at the and causes the to the pocket in the active site by and the of Trp-215 the as the of the active site in the structure of thrombin that the of Trp-215 into the active site is by the environment of the site the catalytic Protein Sci. 1992; PubMed Scopus Google Scholar). of the the in the caused by the of Trp-215 the and changes the orientation of The side of located Å from the site of and the guanidinium group in with Asp-189 in the primary specificity pocket the guanidinium group of Arg-221a a to that of the guanidinium group of the of or of an substrate the of Trp-215 and Arg-221a a conformation of thrombin that is self-inhibited by the of the and active site by and of the of Asp-189 in the primary specificity pocket by and Arg-221a with the and free the side of is to in the Na+-bound fast form The mobility of Arg-187 and the of the the side of Arg-187 to the protein core and the Na+ with the guanidinium group Å from where Na+ The structure of a conformation of thrombin that is to at the Na+ site and at the active in with the of the inactive slow form S* documented by kinetic (10Lai M.T. Di Cera E. Shafer J.A. J. Biol. Chem. 1997; 272: 30275-30282Abstract Full Text Full Text PDF PubMed Scopus (43) Google of the of the structures of with C in and the Na+-bound form with C in (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) the of in the Trp-215 and Arg-221a of a self-inhibited conformation of the enzyme by to and of in the fast form. is the with the water molecules and the network the significant of the with of the interactions with the Arg-187 to its guanidinium group Å from where Na+ in the fast form. The to the of residue Asp-189 and the of the of in the The structure is a of of and and Na+ by the of the thrombin in the free the structure of a single molecule in the asymmetric with molecules in the These the self-inhibited interactions with with and with are on the of Arg-221a and Arg-187 the protein interactions involve the from to but to The structure the active site by Glu-192 in structures of inactive thrombin W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar), of the (12Huntington J.A. Esmon C.T. Structure (Camb.). 2003; 11: 469-479Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar) or significant of the Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). have that the in the of Glu-192 is an of crystal The to of the for have with Na+ binding (9Pineda A.O. Carrell C.J. Bush L.A. Prasad S. Caccia S. Chen Z.W. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: 31842-31853Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). of the is Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and is by the structure of the J. S. and E. Di in where packing interactions differ significantly from in the inactive structure of the Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The of is as in other structures of inactive thrombin W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar) and in the form C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar), to the that of the in is of the of the Na+ site. the of the into the active site is of that in the structures of A.O. Chen Z.W. Caccia S. Savvides S.N. Waksman G. Mathews F.S. Di Cera E. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), W.J. Myles T. Gibbs C.S. Leung L.L. Huntington J.A. J. Biol. Chem. 2004; 279: 26387-26394Abstract Full Text Full Text PDF PubMed Scopus Google Scholar), and inactive C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar). The is to the of Trp-215 causes a in the to C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar). the of Trp-215 into the active site in the structure of be an of the side as in the inactive structure of the Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of Trp-215 and its on the of the inactive slow with the of Arg-221a and Arg-187 documented in thrombin is to that the of the to the inactive slow form and are of the mutation. have that the causes of the network the active site. A that the the inactive slow form from stopped-flow measurements of Na+ binding of Na+ to the is as for and the of the slow phase with of the Na+ of that the has Na+ with the between inactive and active slow forms that is for increases to for the the to the inactive slow form by of the enzyme in the absence of The structure of that thrombin assume an self-inhibited conformation in the absence of as a result of the extreme of the that be from involving inhibitors Protein Sci. 1992; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). The conformation of thrombin binding to the active site and the Na+ site and is the of the inactive slow form S* by stopped-flow kinetic (10Lai M.T. Di Cera E. Shafer J.A. J. Biol. Chem. 1997; 272: 30275-30282Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). the of thrombin to of in of the of at thrombin forms, the Na+-bound fast form the Na+-free slow form and the inactive slow form The of these forms on the Na+ but also on the or to the is also that the S* form may be but may exist as an of as recently suggested C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar), with in by the structures of and Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar). The of Trp-215 in the structure of to the of residue in thrombin specificity and to its documented to between procoagulant and anticoagulant Y.M. Di Cera E. Biochemistry. 2000; PubMed Scopus (47) Google Scholar). The structure and Carrell C.J. A.O. Mathews F.S. C.S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. Bush L.A. Mathews F.S. Di Cera E. Chem. 2006; PubMed Scopus Google Scholar, Huntington J.A. J. 2005; PubMed Scopus Google Scholar) that the high mobility of the side of Trp-215 in the binding site may of of Trp-215 also explain residue is a Na+ binding to thrombin Y.M. Di Cera E. Biochemistry. 2000; PubMed Scopus (47) Google Scholar). the of a at in an to the environment its the W215P mutation in a significant of protein C activation in the presence of of but no effect on the cleavage of or The also shows Na+ activation with for of the Na+ the mutation fibrinogen cleavage significantly protein C activation Y.M. Di Cera E. Biochemistry. 2000; PubMed Scopus (47) Google Scholar), thrombin specificity be by single of residue with the for fibrinogen cleavage to protein C activation for the W215P and for the to of fibrinogen depends on the of the of but of protein C of kcat/Km of and of residue the presence of and of protein C in the absence of was for the and W215P of fibrinogen cleavage as to protein C the presence of and of protein C in the absence of was for the Y.M. Di Cera E. Biochemistry. 2000; PubMed Scopus (47) Google Scholar, Bush L.A. A.O. Caccia S. Di Cera E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and W215P of fibrinogen cleavage as to protein C in a