Mechanism of the Ca2+-induced Enhancement of the Intrinsic Factor VIIa Activity

Abstract

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca2+ binding to a loop (residues 210–220) in the protease domain. Structural analysis revealed that Ca2+ may enhance the activity by attenuating electrostatic repulsion of Glu296 and/or by facilitating interactions between the loop and Lys161 in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys161-interactive residue Asp217 or Asp219 to Ala reduced the amidolytic activity by 40–50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca2+-binding loop appears to suffice for some residual interaction with Lys161, whereas the more severe effect upon replacement of Lys161 is due to abrogation of the interaction with the N-terminal tail. However, Ca2+ attenuation of the repulsion between Asp212 and Glu296 keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp212 in the Ca2+-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca2+-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIaD212N and FVIIaV158D/E296V/M298Q revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca2+-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca2+ stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization. The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca2+ binding to a loop (residues 210–220) in the protease domain. Structural analysis revealed that Ca2+ may enhance the activity by attenuating electrostatic repulsion of Glu296 and/or by facilitating interactions between the loop and Lys161 in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys161-interactive residue Asp217 or Asp219 to Ala reduced the amidolytic activity by 40–50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca2+-binding loop appears to suffice for some residual interaction with Lys161, whereas the more severe effect upon replacement of Lys161 is due to abrogation of the interaction with the N-terminal tail. However, Ca2+ attenuation of the repulsion between Asp212 and Glu296 keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp212 in the Ca2+-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca2+-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIaD212N and FVIIaV158D/E296V/M298Q revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca2+-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca2+ stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization. The proteolytically activated form of blood coagulation factor VII (FVII), 2The abbreviations used are: FVII/VIIacoagulation factor VII/VIIaTFtissue factorATantithrombinsTF-(1–209)soluble TF, residues 1–209Zbenzyloxycarbonyl. FVIIa, behaves zymogen-like with low enzymatic activity as opposed to other, constitutively active serine proteases of the coagulation cascade. Only upon full activation of FVIIa by tissue factor (TF) binding is the cascade initiated by activation of coagulation factors IX and X, in turn leading to the generation of thrombin (1Østerud B. Rapaport S.I. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5260-5264Crossref PubMed Scopus (598) Google Scholar, 2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1635) Google Scholar, 3Walsh P.N. Thromb. Haemostasis. 2001; 86: 75-82Crossref PubMed Scopus (92) Google Scholar). Thrombin then cleaves fibrinogen to fibrin that polymerizes to give a clot that permanently cessates the bleeding (4Kalafatis M. Egan J.O. van't Veer C. Cawthern K.M. Mann K.G. Crit. Rev. Eukaryotic Gene Expression. 1997; 7: 241-280Crossref PubMed Scopus (114) Google Scholar, 5Torbet J. Thromb. Haemostasis. 1995; 73: 785-792Crossref PubMed Scopus (25) Google Scholar). coagulation factor VII/VIIa tissue factor antithrombin soluble TF, residues 1–209 benzyloxycarbonyl. As for all trypsin-like serine proteases, the activation of enzymes in the coagulation cascade involves tethering of the N terminus, created after proteolytic cleavage of the zymogen forms, to the activation domain within the protease domain with concomitant stabilization of its three activation loops (6Brünger A.T. Huber R. Karplus M. Biochemistry. 1987; 26: 5153-5162Crossref PubMed Scopus (55) Google Scholar, 7Huber R. Bode W. Acc. Chem. Res. 1978; 11: 114-122Crossref Scopus (613) Google Scholar, 8Bode W. Huber R. FEBS Lett. 1976; 68: 231-236Crossref PubMed Scopus (123) Google Scholar). In the active conformation of FVIIa, a salt bridge is formed between the N-terminal α-amino group of Ile153 and the side chain of Asp343 in the activation pocket of the activation domain. 3Amino acid sequence numbering (FVIIa, chymotrypsinogen) is as follows: 153, 16; 156, 19; 157, 20; 158, 21; 161, 24; 210, 70; 211, 71; 212, 72; 213, 73; 214, 74; 217, 77; 219, 79; 220, 80; 283, 140; 295, 153; 296, 154; 297, 155, 298, 156; 306, 164; 312, 170; 343, 194. The latter is positioned next to the active site Ser344 (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (689) Google Scholar). FVIIa is only fully active upon TF binding and Ca2+ binding to nine sites located in three different domains, including the Ca2+-binding Glu210–Glu220 loop in the protease domain (10Wildgoose P. Foster D. Schiødt J. Wiberg F.C. Birktoft J.J. Petersen L.C. Biochemistry. 1993; 32: 114-120Crossref PubMed Scopus (61) Google Scholar, 11Sabharwal A.K. Birktoft J.J. Gorka J. Wildgoose P. Petersen L.C. Bajaj S.P. J. Biol. Chem. 1995; 270: 15523-15530Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Upon binding of the cofactors, an activity increase of several 1,000-fold is observed, in part by shifting the equilibrium from the zymogen-like form toward the catalytically active conformation of FVIIa (12Bom V.J.J. Bertina R.M. Biochem. J. 1990; 265: 327-336Crossref PubMed Scopus (168) Google Scholar, 13Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (153) Google Scholar). Ca2+ binding alone leads to a more than 10-fold increase in activity (14Persson E. Petersen L.C. Eur. J. Biochem. 1995; 234: 293-300Crossref PubMed Scopus (32) Google Scholar). Interestingly, the activating effect of TF has been partially mimicked by means of grafting (see Ref. 15Soejima K. Mizuguchi J. Yuguchi M. Nakagaki T. Higashi S. Iwanaga S. J. Biol. Chem. 2001; 276: 17229-17235Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and site-directed mutagenesis (see Ref. 16Persson E. Bak H. Østergaard A. Olsen O.H. Biochem. J. 2004; 379: 497-503Crossref PubMed Scopus (42) Google Scholar) of the loop succeeding the TF-interactive helix (residues 306–312) yielding FVIIa variants with increased intrinsic activity. Replacements of residues in the vicinity of the activation domain have also improved FVIIa activity. Notably, the residues Val158, Glu296, and Met298 appear to form a zymogenicity-determining triad close to the activation domain. Replacements by the corresponding three residues in homologous coagulation factors have resulted in FVIIa variants with substantially increased intrinsic activity (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar, 18Petrovan R.J. Ruf W. J. Biol. Chem. 2001; 276: 6616-6620Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19Petrovan R.J. Ruf W. Biochemistry. 2002; 41: 9302-9309Crossref PubMed Scopus (27) Google Scholar). The most dramatic increase requires conversion of the entire 3-residue motif to that found in thrombin and factor IXa (V158D/E296V/M298Q-FVIIa) (FVIIaDVQ) (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar). Mutation of Glu296 to Val reduced the dependence on Ca2+ suggesting a link to the stimulatory Ca2+ effect, possibly via insertion of the N terminus (20Persson E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google Scholar). Moreover, residue 296 has been linked to macromolecular substrate-induced maturation of the catalytic cleft as inferred from a conformational change upon binding of a monoclonal antibody (21Dickinson C.D. Shobe J. Ruf W. J. Mol. Biol. 1998; 277: 959-971Crossref PubMed Scopus (52) Google Scholar, 22Shobe J. Dickinson C.D. Ruf W. Biochemistry. 1999; 38: 2745-2751Crossref PubMed Scopus (32) Google Scholar). Using a change-of-function mutagenesis and crystal structural approach, we have further investigated the zymogenicity and Ca2+ dependence of FVIIa. We hypothesized that the effects of Ca2+ on FVIIa activity are through stabilization of the Ca2+-binding loop, i.e. stabilization of the interaction(s) of Asp217/Asp219 with Lys161 in the N-terminal tail, and through alleviation of charge repulsion between the loop, primarily Asp212, and the nearby Glu296. The latter residue had been mutated in an earlier study (20Persson E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google Scholar) and the other four were in the study to a of variants to the Lys161, and Asp219 were by Ala to the new interactions or hydrogen Asp212 by the charge with data suggest that Ca2+ binding to the loop in the protease domain of FVIIa the intrinsic activity through stabilization of electrostatic interactions of Asp217 and Asp219 with Lys161 in the N-terminal tail and of the repulsion by Asp212 of Glu296 located close to loop of the activation domain. Asp212 as a new zymogenicity the charge removal to an increased intrinsic activity and of N terminus of the E. FEBS Lett. 1996; PubMed Scopus Google Scholar). The mutations were the and the with in and the were used to the C. and the FVIIa were as C. Thromb. Haemostasis. 98: PubMed Scopus (27) Google Scholar). The the (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar). of and from as (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar, S. M. T. Pedersen A. U. Biochemistry. PubMed Scopus Google Scholar) an and of zymogen either by or by the of or soluble The latter from FVIIa an FVIIa were from FVIIa were by an amidolytic activity the of the FVIIa to and with were with those of of and FVIIa the activity of FVIIa had been the of the were the of FVIIa the active and the active by the activity FVIIa for the of and were from FVIIa variants were by with the as E. Kjalke M. M. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). TF residues as Olsen O.H. E. Sci. 1996; PubMed Scopus Google Scholar). from and conformational amidolytic proteolytic antithrombin from Ref. from Ref. E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google in a new and were in and The amidolytic activity of FVIIa with or were from the for with in a The proteolytic were by FVIIa with for the with and the activity by The of by a FVIIa by the with FVIIa to the activity. of Ile153 by of to FVIIa with in (see were different and 10-fold in and for as for the amidolytic by of FVIIa with and in were different and in and for FVIIa in with were a of and were the and a of E. R. A. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). The as with to Crystal and data the Y. K. T. A. J. A. J. 7: PubMed Scopus Google Scholar). were in a of with were with the W. J. 1993; 26: Scopus Google Scholar). The structures were by the replacement the part of the A. A. J. 1997; 30: Scopus Google Scholar, Biol. PubMed Scopus Google and as a (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (689) Google Scholar). the P. K. Biol. 2004; PubMed Scopus Google Scholar) and as a the part of the Biol. PubMed Scopus Google Scholar, E. P. M. E. Biol. PubMed Scopus Google Scholar). The The Scholar) used for analysis and of intrinsic amidolytic were for and Ala Lys161, and Asp219 the is that of the Asp residues 217, and are side chain Ca2+ (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (689) Google Scholar). The amidolytic activity of FVIIaD212N increased with FVIIa, whereas that of as (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar, E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google Scholar). The three Ala reduced amidolytic In the of in activity between and FVIIa be observed, that the effects of the mutations are only in the of the In and a in amidolytic activity to or further the variants with increased intrinsic we the in the of have that TF the to by with the form of FVIIa as a of maturation of the active site S. Mann K.G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Rapaport S.I. 1993; PubMed Google Scholar). increased of FVIIa variants with increased intrinsic have also been to with the of intrinsic amidolytic activity (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar, E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google Scholar). In with FVIIaD212N and and with and the that the acid in the Ca2+-binding loop and those close to the activation domain the N terminus the of maturation of the active the of FVIIaD212N and from that of FVIIa The proteolytic activity of FVIIaD212N with FVIIa We as an effect, in the of has been to have an proteolytic activity C.D. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). The three Ala of proteolytic activity in with the amidolytic has been that the activating effect of TF on FVIIa is linked to insertion of the N terminus the activation pocket S. H. K. Iwanaga S. J. Biol. Chem. 269: Full Text PDF PubMed Google Scholar). of the α-amino group of the Ile153 of FVIIa of the salt bridge with We used to the of of the N terminus in the activation pocket and found that FVIIaD212N and were about and 10-fold than FVIIa, a more N terminus in variants and that the conformational of FVIIaD212N and are toward the catalytically active conformation with that of FVIIa. We have that Ca2+ has a reduced on the amidolytic activity of due to the E296V mutation (20Persson E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google Scholar). of Ca2+ resulted in the of of the activity of whereas the amidolytic of all the other including and FVIIa were reduced by more than the for the stimulatory effect of Ca2+ on FVIIa we the of the interactions between Asp217 and Asp219 in the Ca2+-binding loop and Lys161 in the N-terminal tail. either Asp217 or Asp219 by Ala reduced the amidolytic activity by in the of Ca2+ The of Lys161 for Ala reduced the activity further residual The reduced activity of the Ala from FVIIa in of and and in FVIIa. The of the Asp to Ala that one Asp residue for some residual interaction with Lys161, whereas the more severe effect upon of Ala for Lys161 is due to abrogation of the interaction of the Asp residues in the Ca2+-binding loop with the N-terminal tail. However, the activity of in the of Ca2+ than that of FVIIa in the of Ca2+ the repulsion between Asp212 and Glu296 by of FVIIaD212N and in with structures of active FVIIaD212N and in with were to and In the of the of in the the to be to the an of primarily due to low of the in these for data above data and of of and are the of the and the corresponding for and are the and factor for is a of from of of bond and are the of the and the corresponding for and are the and factor for is a of from in a new The structures were by means of the replacement acid of the as a (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (689) Google Scholar). in the replacement for of the of and FVIIaD212N with one and were and by several of In the of used with for chain and side chain The were the and further of the by of The mutated residues were in the of or a N-terminal in FVIIaD212N and of the chain and several loops of the domain of TF in the and were in the Altogether, of and of residues in were in a of and in these is to be the for or mutations were located in the protease from the and appear to the of the protease domain as The protease of variants an also in the domain as from The of and the FVIIa variants and the of the and of the FVIIa variants were to the of and (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (689) Google Scholar). However, be between the FVIIa variants in the of the mutated residues in the protease domain. In the of the three mutated and are in the a of new hydrogen bond is by the introduction of the side and involving the of and a is in with a (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar). hydrogen bond appears to a effect on the N terminus in with the of the α-amino group of Ile153 and The is with a low factor of with the of for all in the In the the has a factor of with an for all of that the is in stabilization of the activation pocket of in a more Structural in the binding including activation loop of the activation are all in with as from is located between mutated residues and Asp212 positioned in the Ca2+-binding loop the side chain of Asp212 is more toward the side chain of with Glu296 is a electrostatic repulsion between a residue in the Ca2+-binding loop and a residue succeeding activation loop is by in hydrogen bond in the between in the Ca2+-binding loop and Glu296 as by the E296V the Ca2+-binding loop side chain interactions with the Met298 In the of residue in a conformation only different from that of residue Asp212 hydrogen bond the mutated interaction with the as as other more of the loop were and of in hydrogen binding of Glu296 and be hydrogen bond between these residues the mutation the charge between side and 296, be with due to the of the The conformation of the Ca2+-binding loop appears to the activation domain and to the active is to that the chain hydrogen bonds to and to tethering the Ca2+ loop to the 296 were only in the FVIIaD212N and structures with the FVIIa The mutated residue appeared to residues further in the Ca2+-binding loop, including residues and as as Lys161 positioned in the N-terminal tail and for In we have investigated the zymogenicity of FVIIa and factors that the insertion of the N We have residue Asp212, located in the Ca2+-binding loop of the protease as for the zymogen-like of FVIIa, the FVIIaD212N leads to a more N terminus and a more active site as from and more these four residues the conformational equilibrium toward the catalytically form of FVIIa. In structures of coagulation thrombin and these four are by that support hydrogen bond and we that be FVIIa with improved catalytic as the an earlier that the intrinsic activity of with increased tethering of the N-terminal tail and more insertion of the N terminus (17Persson E. Kjalke M. Olsen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13583-13588Crossref PubMed Scopus (115) Google Scholar, E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google by a hydrogen bond by the mutations and Only FVIIa has an Asp residue in to 212, whereas and all have in In the constitutively active and in corresponding to and 296, in FVIIa, a hydrogen bond to that in the FVIIaD212N Interestingly, an effect on activation for FVIIaD212N be observed, is in with the proteolytic activity of in the of C.D. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). that only is Asp212 a zymogenicity-determining residue in FVIIa, that also is part of an located in the Ca2+-binding We have that mutation E296V the Ca2+ dependence of FVIIa, whereas the the of the N terminus, the maturation of the active and the amidolytic activity (20Persson E. Olsen O.H. Eur. J. Biochem. 2002; 269: 5950-5955Crossref PubMed Scopus (19) Google Scholar). As in the mutation E296V is responsible for the dependence by side chain interactions with the Ca2+-binding loop that activation loop The of Glu296 appears linked to Asp212 via charge has also been that Glu296 is linked to maturation of the catalytic cleft an antibody (21Dickinson C.D. Shobe J. Ruf W. J. Mol. Biol. 1998; 277: 959-971Crossref PubMed Scopus (52) Google Scholar, 22Shobe J. Dickinson C.D. Ruf W. Biochemistry. 1999; 38: 2745-2751Crossref PubMed Scopus (32) Google Scholar) effect abolished by the Moreover, binding to the active site of FVIIa appears to a conformational change of the Glu296 side we a zymogen-like FVIIa with and Ca2+ the conformational of Glu296 and Asp212 with the activation domain and an Glu210–Glu220 loop are However, the of the residues the between the Ca2+-binding loop and the of activation loop N-terminal loop is a part of the activation domain of trypsin-like proteases and from a to a conformation upon activation R. Bode W. Acc. Chem. Res. 1978; 11: 114-122Crossref Scopus (613) Google Scholar, 8Bode W. Huber R. FEBS Lett. 1976; 68: 231-236Crossref PubMed Scopus (123) Google Scholar). loop in FVIIa has been to upon TF binding as from of Olsen O.H. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Olsen O.H. Østergaard H. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues to have a reduced upon binding to TF to of Olsen O.H. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the an TF to that for Olsen O.H. Østergaard H. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that charge removal in 296 the hydrogen bond interactions leading to reduced Ca2+ dependence and activity. of Ca2+ the activity of FVIIa to than (14Persson E. Petersen L.C. Eur. J. Biochem. 1995; 234: 293-300Crossref PubMed Scopus (32) Google the and effects of a Ca2+ in the Glu210–Glu220 D212N and E296V further the electrostatic The Ca2+ effect is for FVIIa as with than the effect of Interestingly, FVIIa has TF, Ca2+ appears to have its stimulatory and only as a T. A. H. J. Biochem. 1995; PubMed Scopus Google Scholar). Ca2+ binding to stimulates the amidolytic activity by is than for FVIIa T. F.C. Thromb. Res. Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 269: Full Text PDF PubMed Google Scholar). is also and for FVIIa, been to be by insertion of the N terminus the activation pocket A. S. Bajaj S.P. J. Mol. Biol. PubMed Scopus Google Scholar). The of appears to with the charge of the Ca2+-binding loop and the of the side chain in to in FVIIa. The in in FVIIa hydrogen bond between Asp217 and Asp219 in the Ca2+-binding loop and Lys161 in the N-terminal tail. Lys161 the N-terminal tail to the Ca2+-binding loop and appears to be in the maturation of the active site of FVIIa. Lys161 to Ala in a in proteolytic S. H. K. Iwanaga S. J. Biol. Chem. 269: Full Text PDF PubMed Google Scholar) and amidolytic activity as The effects of either Asp217 or Asp219 were that some interaction with Lys161 is The effect of the K161A mutation is to the entire effect of FVIIa of The FVIIaD212N Ca2+ that the altered interactions between the Ca2+-binding loop and Lys161 and Glu296 in the are responsible for the Ca2+ The zymogen-like conformation of FVIIa is and for of the blood coagulation cascade only upon binding to TF the site of Val158, Asp212, Glu296, and Met298 are fully responsible Notably, the four residues are between FVIIa from different In we have and the variants and conformation of the form is toward the active with increased intrinsic as the the crystal structures were with enzymes to a to the of FVIIa, is be with However, we that the in the activation are to the TF binding site and the active are with the of FVIIa in with and be the conformational of the catalytically of and is by the of hydrogen of and N-terminal in FVIIa and upon TF that the structural between the FVIIa in and the activation domain appear to be in the of TF Olsen O.H. Østergaard H. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the structures that new hydrogen bond increase the between the loop activation loop the Ca2+-binding Glu210–Glu220 loop, and the N-terminal tail. are for of the of FVIIa. We B. for of the and Østergaard and for