Role of sequence and position of the cleavage sites in prothrombin activation
2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.100955
ISSN1083-351X
AutoresBosko M. Stojanovski, Enrico Di,
Tópico(s)Vitamin K Research Studies
ResumoIn the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences 261DEDSDRAIEGRTATSEYQT279 and 310RELLESYIDGRIVEGSDAE328 were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation. In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences 261DEDSDRAIEGRTATSEYQT279 and 310RELLESYIDGRIVEGSDAE328 were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation. Prothrombin is a multidomain vitamin-K-dependent zymogen that is converted to the active enzyme thrombin in the penultimate step of the coagulation cascade (1Davie E.W. Fujikawa K. Kisiel W. The coagulation cascade: Initiation, maintenance, and regulation.Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1588) Google Scholar). The multidomain assembly comprises an N-terminal Gla domain (residues 1–46), kringle 1 (residues 65–143), kringle 2 (residues 170–248), and a C-terminal protease domain (residues 285–579) connected by three intervening linkers (Fig. 1A) (2Chinnaraj M. Chen Z. Pelc L.A. Grese Z. Bystranowska D. Di Cera E. Pozzi N. Structure of prothrombin in the closed form reveals new details on the mechanism of activation.Sci. Rep. 2018; 8: 2945Crossref PubMed Scopus (12) Google Scholar, 3Pozzi N. Chen Z. Di Cera E. How the linker connecting the two kringles influences activation and conformational plasticity of prothrombin.J. Biol. Chem. 2016; 291: 6071-6082Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 4Pozzi N. Chen Z. Gohara D.W. Niu W. Heyduk T. Di Cera E. Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation.J. Biol. Chem. 2013; 288: 22734-22744Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 5Pozzi N. Chen Z. Pelc L.A. Shropshire D.B. Di Cera E. The linker connecting the two kringles plays a key role in prothrombin activation.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 7630-7635Crossref PubMed Scopus (31) Google Scholar). Thrombin formation requires cleavage of prothrombin at two sites, R271 and R320, by the prothrombinase complex composed of factor Xa (fXa), cofactor Va (fVa), and phospholipids (6Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 7Mann K.G. Thrombin formation.Chest. 2003; 124: 4S-10SAbstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 8Orcutt S.J. Krishnaswamy S. Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin.J. Biol. Chem. 2004; 279: 54927-54936Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Cleavage at R271 sheds the Gla domain and two kringles and generates the inactive intermediate prethrombin-2 (Fig. 1A). The alternative cleavage at R320 separates the A and B chains of the protease domain that remain connected through a disulfide bond and generates the active intermediate meizothrombin (Fig. 1A). The presence of fVa directs activation along the meizothrombin pathway and greatly (>1000-fold) accelerates the rate of cleavage at R320, but has a smaller effect (30-fold) on the cleavage of meizothrombin at R271 (9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Meizothrombin accumulates as an intermediate when prothrombinase is assembled on the membrane of red blood cells (10Whelihan M.F. Zachary V. Orfeo T. Mann K.G. Prothrombin activation in blood coagulation: The erythrocyte contribution to thrombin generation.Blood. 2012; 120: 3837-3845Crossref PubMed Scopus (94) Google Scholar), synthetic liposomes (6Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 11Lentz B.R. Exposure of platelet membrane phosphatidylserine regulates blood coagulation.Prog. Lipid Res. 2003; 42: 423-438Crossref PubMed Scopus (242) Google Scholar, 12Tans G. Janssen-Claessen T. Hemker H.C. Zwaal R.F. Rosing J. Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma.J. Biol. Chem. 1991; 266: 21864-21873Abstract Full Text PDF PubMed Google Scholar), and microparticles (13Nomura S. Shimizu M. Clinical significance of procoagulant microparticles.J. Intensive Care. 2015; 3: 2Crossref PubMed Scopus (99) Google Scholar). On the other hand, platelets direct prothrombinase to cleave initially at R271 along the prethrombin-2 pathway (14Haynes L.M. Bouchard B.A. Tracy P.B. Mann K.G. Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism.J. Biol. Chem. 2012; 287: 38647-38655Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Wood J.P. Silveira J.R. Maille N.M. Haynes L.M. Tracy P.B. Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.Blood. 2011; 117: 1710-1718Crossref PubMed Scopus (38) Google Scholar), which is also preferred in the absence of fVa (6Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 7Mann K.G. Thrombin formation.Chest. 2003; 124: 4S-10SAbstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Although meizothrombin and prothrombin share the same multidomain assembly, there are significant differences in the structure of the two proteins. Cleavage at R320 triggers the Huber–Bode mechanism of zymogen activation (16Huber R. Bode W. Structural basis of the activation and action of trypsin.Acc. Chem. Res. 1978; 11: 114-122Crossref Scopus (601) Google Scholar) where a new N-terminus engages a conserved Asp residue next to the catalytic Ser and organizes the architecture of the active site and primary specificity pocket (17Chen Z. Pelc L.A. Di Cera E. Crystal structure of prethrombin-1.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 19278-19283Crossref PubMed Scopus (30) Google Scholar, 18Martin P.D. Malkowski M.G. Box J. Esmon C.T. Edwards B.F. New insights into the regulation of the blood clotting cascade derived from the X-ray crystal structure of bovine meizothrombin des F1 in complex with PPACK.Structure. 1997; 5: 1681-1693Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Papaconstantinou M.E. Gandhi P.S. Chen Z. Bah A. Di Cera E. Na+ binding to meizothrombin desF1.Cell Mol. Life Sci. 2008; 65: 3688-3697Crossref PubMed Scopus (26) Google Scholar), along with exosite I (20Anderson P.J. Bock P.E. Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin.J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), the Na+-binding site (19Papaconstantinou M.E. Gandhi P.S. Chen Z. Bah A. Di Cera E. Na+ binding to meizothrombin desF1.Cell Mol. Life Sci. 2008; 65: 3688-3697Crossref PubMed Scopus (26) Google Scholar, 21Kroh H.K. Tans G. Nicolaes G.A. Rosing J. Bock P.E. Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation.J. Biol. Chem. 2007; 282: 16095-16104Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and the autolysis loop (22Acquasaliente L. Pelc L.A. Di Cera E. Probing prothrombin structure by limited proteolysis.Sci. Rep. 2019; 9: 6125Crossref PubMed Scopus (4) Google Scholar). Activation also shifts the predominant closed conformation of prothrombin to the open form of meizothrombin (2Chinnaraj M. Chen Z. Pelc L.A. Grese Z. Bystranowska D. Di Cera E. Pozzi N. Structure of prothrombin in the closed form reveals new details on the mechanism of activation.Sci. Rep. 2018; 8: 2945Crossref PubMed Scopus (12) Google Scholar, 23Pozzi N. Bystranowska D. Zuo X. Di Cera E. Structural architecture of prothrombin in solution revealed by single molecule spectroscopy.J. Biol. Chem. 2016; 291: 18107-18116Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 24Stojanovski B.M. Pelc L.A. Zuo X. Pozzi N. Di Cera E. Enhancing the anticoagulant profile of meizothrombin.Biomol. Concepts. 2018; 9: 169-175Crossref PubMed Scopus (4) Google Scholar, 25Chen Q. Lentz B.R. Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin.Biochemistry. 1997; 36: 4701-4711Crossref PubMed Scopus (31) Google Scholar). The shift affects cleavage at R271 by either fXa or prothrombinase, which is considerably faster in meizothrombin (8Orcutt S.J. Krishnaswamy S. Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin.J. Biol. Chem. 2004; 279: 54927-54936Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Prothrombin mutants stabilized in the open form are activated by prothrombinase with accumulation of the prethrombin-2 intermediate because of increased specificity for the R271 site (2Chinnaraj M. Chen Z. Pelc L.A. Grese Z. Bystranowska D. Di Cera E. Pozzi N. Structure of prothrombin in the closed form reveals new details on the mechanism of activation.Sci. Rep. 2018; 8: 2945Crossref PubMed Scopus (12) Google Scholar). Binding of substrates to the active site of prothrombin has been reported to enhance cleavage at R271 (26Bianchini E.P. Orcutt S.J. Panizzi P. Bock P.E. Krishnaswamy S. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10099-10104Crossref PubMed Scopus (49) Google Scholar, 27Kroh H.K. Panizzi P. Tchaikovski S. Baird T.R. Wei N. Krishnaswamy S. Tans G. Rosing J. Furie B. Furie B.C. Bock P.E. Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo.J. Biol. Chem. 2011; 286: 23345-23356Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 28Chakraborty P. Acquasaliente L. Pelc L.A. Di Cera E. Interplay between conformational selection and zymogen activation.Sci. Rep. 2018; 8: 4080Crossref PubMed Scopus (11) Google Scholar), presumably because active site occupancy stabilizes the open form in both prothrombin and meizothrombin (2Chinnaraj M. Chen Z. Pelc L.A. Grese Z. Bystranowska D. Di Cera E. Pozzi N. Structure of prothrombin in the closed form reveals new details on the mechanism of activation.Sci. Rep. 2018; 8: 2945Crossref PubMed Scopus (12) Google Scholar, 23Pozzi N. Bystranowska D. Zuo X. Di Cera E. Structural architecture of prothrombin in solution revealed by single molecule spectroscopy.J. Biol. Chem. 2016; 291: 18107-18116Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 24Stojanovski B.M. Pelc L.A. Zuo X. Pozzi N. Di Cera E. Enhancing the anticoagulant profile of meizothrombin.Biomol. Concepts. 2018; 9: 169-175Crossref PubMed Scopus (4) Google Scholar, 25Chen Q. Lentz B.R. Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin.Biochemistry. 1997; 36: 4701-4711Crossref PubMed Scopus (31) Google Scholar). On the other hand, mutations that interfere with the zymogen-to-protease transition and the concomitant shift from the closed to the open form (22Acquasaliente L. Pelc L.A. Di Cera E. Probing prothrombin structure by limited proteolysis.Sci. Rep. 2019; 9: 6125Crossref PubMed Scopus (4) Google Scholar) decrease the rate of cleavage at R271 (26Bianchini E.P. Orcutt S.J. Panizzi P. Bock P.E. Krishnaswamy S. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10099-10104Crossref PubMed Scopus (49) Google Scholar). Because fXa preferentially cleaves at R271 and prothrombinase preferentially cleaves at R320 (8Orcutt S.J. Krishnaswamy S. Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin.J. Biol. Chem. 2004; 279: 54927-54936Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), it is important to establish whether the preference is determined by the conformation of the protein and/or the specific sequence around the cleavage sites at R271 and R320. About 79% of residues in the P11–P8′ positions (29Schechter I. Berger A. On the size of the active site in proteases. I. Papain.Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4641) Google Scholar) of the 261DEDSDRAIEGRTATSEYQT279 and 310RELLESYIDGRIVEGSDAE328 sequences are not conserved between the two cleavage sites (Fig. 1B), especially in the primed region (C-terminally of the P1 Arg). In the nonprimed region (N-terminally of the P1 Arg), four out of 11 residues are conserved at both cleavage sites, including the P1 Arg, P2 Gly, P4 Ile, and P10 Glu (Fig. 1B). Previous studies have addressed how the sequence affects cleavage at R320 and shown that replacements at the P1–P3 positions in prethrombin-2 adversely affect formation of thrombin (30Orcutt S.J. Pietropaolo C. Krishnaswamy S. Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.J. Biol. Chem. 2002; 277: 46191-46196Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, the P1 and P2 residues are conserved between the R271 and R320 sites, and the P3 residue is acidic in both cases (Fig. 1B). The P1′–P3′ residues at the R320 site have also been mutated and shown to have a very small effect on the activation rate (26Bianchini E.P. Orcutt S.J. Panizzi P. Bock P.E. Krishnaswamy S. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10099-10104Crossref PubMed Scopus (49) Google Scholar). Overall, these previous studies have offered little insight into the molecular origin of preferential cleavage at the R271 and R320 sites. Differences in the extended sequences at the two sites turned out to be more informative, as shown below. The prothrombin loop-swap mutants proT320/320 and proT271/271 were engineered to examine how the pathway of activation is affected by the amino acid sequence at the R271 and R320 cleavage sites. proT320/320 has the entire P11–P8′ sequence at the R271 site replaced with that of the R320 site and vice versa for proT271/271 (Fig. 1B). Prothrombin is activated by fXa in the absence of fVa with initial cleavage at R271 and formation of prethrombin-2, followed by cleavage at R320 and appearance of thrombin (Fig. 2). The lack of detectable levels of meizothrombin indicates that the two scissile bonds are attacked sequentially, with the proteolysis at R271 always preceding that at R320. If the sequential cleavage is caused by differences in the amino acid sequence at the two sites of prothrombin, then replacing the sequence at the R320 site with that of the R271 site should accelerate cleavage by fXa at the swapped region in proT271/271 and give evidence of activation also along the meizothrombin pathway. Indeed, proT271/271 is activated by fXa along both pathways. Activation along the meizothrombin pathway is evident from the appearance of the fragment 1.2.A band during the early time course of the reaction, which denotes initial cleavage at R320, while accumulation of prethrombin-2 documents the additional cleavage of proT271/271 at the R271 site (Fig. 2). Characterization of the activation pathway of proT320/320 provides further evidence that the sequence makes an important contribution in directing the sequential cleavage by fXa. proT320/320 is activated by fXa along the prethrombin-2 pathway, but the initial cleavage at R271 is significantly slower and the appearance of the prethrombin-2 band is delayed compared with wild-type (Figs. 2 and 3). The slower rate of cleavage implies that fXa has a strong preference for the native sequence at the R271 site. Initial cleavage at R155 leads to formation of the zymogen prethrombin-1 composed of the kringle 2 and protease domain (Fig. 1A). The prethrombin-1 band was also detected for all prothrombin variants, with the intensity being most pronounced for proT320/320 (Fig. 2) due to slower cleavage at the 271 site. The appearance of prethrombin-1 is not due to cleavage by thrombin because the reactions were carried out in the presence of DAPA (a selective thrombin inhibitor). Furthermore, formation of prethrombin-1 was also detected with the inactive prothrombin mutant S525A, where the catalytic Ser is replaced by Ala, proving that cleavage at R155 is catalyzed by fXa (5Pozzi N. Chen Z. Pelc L.A. Shropshire D.B. Di Cera E. The linker connecting the two kringles plays a key role in prothrombin activation.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 7630-7635Crossref PubMed Scopus (31) Google Scholar).Figure 3Intermediates of prothrombin activation. Optical densitometry of the SDS-PAGE gels shown in Figure 2 using ImageJ quantifies the intensities of the bands of (A) prothrombin and (B) F1.2 A in the presence of fVa, and of (C) prothrombin and (D) prethrombin-2 in the absence of fVa. Data points refer to wild-type (black), proT320/320 (purple), and proT271/271 (gray). Accumulation of F1.2 A (B) for activation of proT320/320 and proT271/271 by prothrombinase is due to slower cleavage at R271. The slower consumption of prothrombin (C) and the delayed appearance of prethrombin-2 (D) for activation of proT320/320 by fXa are also due to slower cleavage at R271.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we examined the pathway of activation in the presence of fVa. The prothrombinase complex activates prothrombin along the meizothrombin pathway documented by the transient appearance of fragment F1.2.A during the early stages of the reaction (Figs. 2 and 3). Disappearance of this band signals subsequent cleavage at R271 and formation of thrombin (Fig. 2). If sequential cleavage is caused by differences in the amino acid sequence at the two sites of prothrombin, then replacing the sequence at the R271 site with that of the R320 site should accelerate cleavage by prothrombinase at the swapped site in proT320/320 and shift activation toward the prethrombin-2 pathway. Contrary to this expectation, prothrombinase activates proT320/320 along the meizothrombin pathway with no evidence of prethrombin-2 formation (Fig. 2). The rate of cleavage at the R271 site carrying the sequence of the R320 site is significantly slower, as evidenced by the prolonged accumulation of fragment F1.2.A and the delayed appearance of fragment F1.2 compared to wild-type (Figs. 2 and 3). Importantly, prothrombinase activates proT271/271 along the meizothrombin pathway as well, proving that swapping the R320 sequence with that of the R271 site does not change the pathway of activation (Fig. 2). The R271 site in proT271/271 is cleaved at a slower rate because of the prolonged accumulation of fragment F1.2.A compared with wild-type (Figs. 2 and 3). A similar observation has been reported for a prothrombin mutant with the P1′–P3′ residues at the R320 site replaced by those of the R271 site (26Bianchini E.P. Orcutt S.J. Panizzi P. Bock P.E. Krishnaswamy S. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10099-10104Crossref PubMed Scopus (49) Google Scholar). The likely explanation is that replacement of the P1′–P3′ sites in this and the proT271/271 mutant impairs the transition to the open form of meizothrombin based on correct formation of the I321-D359 H-bond of the Huber–Bode mechanism of activation (16Huber R. Bode W. Structural basis of the activation and action of trypsin.Acc. Chem. Res. 1978; 11: 114-122Crossref Scopus (601) Google Scholar), thereby making the R271 site less susceptible to cleavage. Overall, our data indicate a major difference in preferential cleavage between fXa and prothrombinase, due entirely to the absence/presence of fVa. Preferential cleavage is dictated by the sequence and not the position in the absence of fVa and by the position but not the sequence in the presence of cofactor. Current assays that monitor cleavage at R271 in prothrombin rely upon rates measured by optical densitometry or by monitoring modest differences in the level of DAPA fluorescence due to binding to meizothrombin and thrombin (9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To overcome these limitations and examine how the sequence affects the rate of cleavage at R271, we developed a FRET assay using the mutant S101C/R155A/S478C/S525A, proTRS, where the replacement R155A prevents cleavage at this site, S525A avoids (auto)proteolysis and the two Cys substitutions position an Alexa Fluor 555/647 pair at kringle 1 and the protease domain. Labeling at these sites has no influence on the functional properties of prothrombin (23Pozzi N. Bystranowska D. Zuo X. Di Cera E. Structural architecture of prothrombin in solution revealed by single molecule spectroscopy.J. Biol. Chem. 2016; 291: 18107-18116Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). When the activation of proTRS by fXa was monitored by changes in FRET, the resulting progress curve could be described by a single exponential (Fig. 4A) with a rate constant of 0.53 μM−1s−1 in close agreement with the value of kcat/Km = 0.64 μM−1s−1 reported for cleavage of R271 by fXa (9Brufatto N. Nesheim M.E. Analysis of the kinetics of prothrombin activation and evidence that two equilibrating forms of prothrombinase are involved in the process.J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The loss of FRET coincides with cleavage at R271 and separation of the protease domain from the rest of the protein containing the Gla domain and two kringles (Figs. 1A and 4A). To demonstrate this point, R271 and R320 were replaced individually by Gln. SDS-PAGE confirmed that fXa does not cleave at the R271Q and R320Q sites (data not shown). Progress curves of proTRS,R320Q (cleaved at R271 only) activation by fXa measured by changes in FRET were superimposable to those of proTRS (cleaved at both R271 and R320), but those of proTRS,R271Q (cleaved at R320 only) were drastically slower (Fig. 4A). The slower loss of FRET in proTRS,R271Q is due to a conformational change that coincides with cleavage at R320 and formation of meizothrombin (Fig. 4E). These results demonstrate that activation of proTRS by fXa proceeds along the prethrombin-2 pathway and that the loss of FRET is caused by cleavage at R271. Next we replaced the P11–P8′ residues at the R271 site with those at the R320 site in the proTRS background, proTRS,320/320. Two additional mutants contained replacement of the nonprimed P11–P1 residues, proTRS,320/320np, or the primed P1′–P8′ residues, proTRS,320/320p (Fig. 1B). We verified that the single-molecule FRET (smFRET) profiles of these mutants populate the same compact conformation as wild-type prothrombin (Fig. 4E), ruling out effects on the overall architecture of the protein (data not shown). Activation by fXa monitored by changes in FRET showed a 9-fold reduction in the kcat/Km value for proTRS,320/320 compared with proTRS (Table 1, Fig. 4C), in agreement with a delayed appearance of the prethrombin-2 band by SDS-PAGE (Fig. 2). The loss of specificity in proTRS,320/320 is due mostly to replacement of the nonprimed residues: the kcat/Km value was reduced ninefold for proTRS,320/320np but only marginally for proTRS,320/320p (Table 1, Fig. 4C). The primed and nonprimed portions of the sequence contribute to cleavage by fXa in an additive manner, with a free energy of coupling ΔGc = –0.2 kcal/mol (31Di Cera E. Site-specific analysis of mutational effects in proteins.Adv. Protein Chem. 1998; 51: 59-119Crossref PubMed Google Scholar), underscoring a nearly independent contribution of the two fragments. Because the P1, P2, and P4 residues are conserved between the R271 and R320 sites and the P3 residue is acidic in both cases, our data show that the distal residues that occupy the P5–P11 positions play an important role for the preferential recognition of the R271 site by fXa.Table 1Specificity constants (kcat/Km in μM−1s−1) for cleavage of R271 in ProTRS (by fXa) and MzRS (by prothrombinase) measured by loss of FRETSequence at R271ProTRSMzRSWT0.53 ± 0.02166 ± 2P11–P8′ of the R320 site0.058 ± 0.0029.5 ± 0.1P11–P1 of the R320 site0.061 ± 0.001117 ± 8P1′–P8′ of the R320 site0.33 ± 0.01127 ± 9Experimental conditions: 20 mM Tris, 145 mM NaCl, 5 mM CaCl2, 50 μM phospholipids, 0.1% PEG8000, 0.01% Tween20, pH 7.5 at 20 °C. Open table in a new tab Experimental conditions: 20 mM Tris, 145 mM NaCl, 5 mM CaCl2, 50 μM phospholipids, 0.1% PEG8000, 0.01% Tween20, pH 7.5 at 20 °C. FRET measurements were extended to prothrombin activation by prothrombinase along the meizothrombin pathway triggered by cleava
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