The High Affinity Calcium-binding Sites in the Epidermal Growth Factor Module Region of Vitamin K-dependent Protein S
1997; Elsevier BV; Volume: 272; Issue: 37 Linguagem: Inglês
10.1074/jbc.272.37.23255
ISSN1083-351X
AutoresYvonne Stenberg, Sara Linse, Torbjörn Drakenberg, Johan Stenflo,
Tópico(s)Atrial Fibrillation Management and Outcomes
ResumoVitamin K-dependent protein S, a cofactor of the anticoagulant enzyme-activated protein C, has four epidermal growth factor (EGF)-like modules, all of which have one partially hydroxylated Asp (EGF 1; β-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; β-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5′-Br2BAPTA, we have now determined the Ca2+affinity of recombinant fragments containing EGF modules 1–3, 1–4, 2–3, and 2–4. EGF modules 1–4 and 2–4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10−10 to 10−8m in the absence of salt and from 10−8 to 10−6m in the presence of 0.15 m NaCl. In contrast, in EGF 1–3 and EGF 2–3, the Ca2+ affinity is 2–4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate N-terminal neighbor EGF 3 by a factor of approximately 230. In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a factor of approximately 25. The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1–4 and EGF 2–4 is 103–105-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. This difference is much higher than the approximately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ligands. The structural basis for the difference in Ca2+affinity is not yet understood. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme-activated protein C, has four epidermal growth factor (EGF)-like modules, all of which have one partially hydroxylated Asp (EGF 1; β-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; β-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5′-Br2BAPTA, we have now determined the Ca2+affinity of recombinant fragments containing EGF modules 1–3, 1–4, 2–3, and 2–4. EGF modules 1–4 and 2–4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10−10 to 10−8m in the absence of salt and from 10−8 to 10−6m in the presence of 0.15 m NaCl. In contrast, in EGF 1–3 and EGF 2–3, the Ca2+ affinity is 2–4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate N-terminal neighbor EGF 3 by a factor of approximately 230. In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a factor of approximately 25. The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1–4 and EGF 2–4 is 103–105-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. This difference is much higher than the approximately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ligands. The structural basis for the difference in Ca2+affinity is not yet understood. Protein S is a vitamin K-dependent plasma protein that functions as a cofactor to activated protein C, a regulator of blood coagulation, by enhancing the activated protein C-mediated rates of degradation of factors Va and VIIIa (1Dahlbäck B. Thromb. Haemostasis. 1991; 66: 49-61Crossref PubMed Scopus (323) Google Scholar, 2Dahlbäck B. Stenflo J. Stamatoyannopoulos G. Nienhuis A.W. Majerus P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Co., Philadelphia1994: 599-628Google Scholar, 3Dahlbäck D. Stenflo J. Bloom A.L. Forbes C.D. Thomas D.P. Tuddenham E.G.D. Haemostasis and Thrombosis. 1. Churchill Livingstone, Edinburgh, UK1994: 671-698Google Scholar). Protein S consists of an N-terminal γ-carboxyglutamic acid (Gla) 1The abbreviations used are: Gla, γ-carboxyglutamic acid; 5,5′-Br2BAPTA, 5,5′-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; EGF, epidermal growth factor; EGF 1–3, residues 73–203; EGF 1–4, residues 73–245; EGF 2–3, residues 115–203; and EGF 2–4, residues 115–245, all in human protein S; SF 9 cells, Spodoptera frugiperda ovarian cells. 1The abbreviations used are: Gla, γ-carboxyglutamic acid; 5,5′-Br2BAPTA, 5,5′-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; EGF, epidermal growth factor; EGF 1–3, residues 73–203; EGF 1–4, residues 73–245; EGF 2–3, residues 115–203; and EGF 2–4, residues 115–245, all in human protein S; SF 9 cells, Spodoptera frugiperda ovarian cells.-containing module that is followed by a module with a thrombin-sensitive peptide bond and four epidermal growth factor (EGF)-like modules, whereas the C-terminal half of the molecule is occupied by a module that is homologous to steroid hormone-binding proteins (4Dahlbäck B. Lundwall Å. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4199-4203Crossref PubMed Scopus (106) Google Scholar, 5Lundwall Å. Dackowski W. Cohen E.H. Shaffer M. Mahr A. Dahlbäck B. Stenflo J. Wydro R.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Crossref PubMed Scopus (149) Google Scholar, 6Gershagen S. Fernlund P. Lundwall Å. FEBS Lett. 1987; 220: 129-135Crossref PubMed Scopus (77) Google Scholar, 7Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (328) Google Scholar). Calcium binding to vitamin K-dependent clotting factors, including protein S, is complex, >10 calcium ions being bound to 2 or 3 types of sites that profoundly influence the function of these proteins (8Stenflo J. Dahlbäck B. Stamatoyannopoulos G. Nienhuis A.W. Majerus P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Co., Philadelphia1994: 565-598Google Scholar). The Gla module binds Ca2+ and endows protein S with membrane affinity, whereas the EGF module-containing part of protein S appears to be involved in the interaction with activated protein C (9Dahlbäck B. Hildebrand B. Malm J. J. Biol. Chem. 1990; 265: 8127-8135Abstract Full Text PDF PubMed Google Scholar,10He X. Shen L. Dahlbäck B. Eur. J. Biochem. 1995; 227: 433-440Crossref PubMed Scopus (39) Google Scholar).The EGF modules in protein S are members of a large family of similar modules that are found in extracellular and membrane mosaic proteins in species ranging from Caenorhabditis elegans toDrosophila melanogaster and humans (7Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (328) Google Scholar, 11Appella E. Weber I.T. Blasi F. FEBS Lett. 1988; 231: 1-4Crossref PubMed Scopus (236) Google Scholar, 12Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar). Several of these proteins are involved in cell differentiation, blood coagulation and fibrinolysis, and in the complement and fibrinolytic systems. The EGF modules are approximately 45 amino acids long and contain 6 cysteine residues that are paired in a characteristic manner: 1 to 3, 2 to 4, and 5 to 6, with a double-stranded β-sheet as the main structural feature. EGF modules provide a structural scaffold that supports protein-protein interactions, serves as spacer units, and orients adjacent modules relative to each other in a way that sustains biological activity.EGF modules 2–4 of protein S belong to a large subgroup of such modules that has a Ca2+ binding sequence motif DI/VDE (or variants thereof) before the first Cys residue (13Rees D.J.G. Jones I.M. Handford P.A. Walter S.J. Esnouf M.P. Smith K.J. Brownlee G.G. EMBO J. 1988; 7: 2053-2061Crossref PubMed Scopus (186) Google Scholar, 14Handford P.A. Mayhew M. Baron M. Winship P.R. Campbell I.D. Brownlee G.G. Nature. 1991; 351: 164-167Crossref PubMed Scopus (245) Google Scholar), and the motifXD*/N*XXXXY/FX between the third and fourth Cys residue that is required for hydroxylation of Asp/Asn (denoted by an asterisk in the sequence) toerythro-β-hydroxyaspartic acid orerythro-β-hydroxyasparagine (12Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar, 15Stenflo J. Öhlin A.K. Owen W.G. Schneider W.J. J. Biol. Chem. 1988; 263: 21-24Abstract Full Text PDF PubMed Google Scholar, 16Stenflo J. Lundwall Å. Dahlbäck B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 368-372Crossref PubMed Scopus (116) Google Scholar). The Asp*/Asn* and Tyr/Phe residues are adjacent in the major double-stranded β-sheet. The two sequence motifs appear to be coupled and phylogenetically conserved in Ca2+ binding EGF modules. There are however exceptions, such as the first EGF module of protein S, which contains the hydroxylation motif but lacks the Ca2+ consensus sequence before the first Cys residue.Calcium binding to an EGF module was first observed in protein C and subsequently in factors IX and X (17Öhlin A.K. Landes G. Bourdon P. Oppenheimer C. Wydro R.M. Stenflo J. J. Biol. Chem. 1988; 263: 19240-19248Abstract Full Text PDF PubMed Google Scholar, 18Öhlin A.K. Linse S. Stenflo J. J. Biol. Chem. 1988; 263: 7411-7417Abstract Full Text PDF PubMed Google Scholar, 19Persson E. Selander M. Linse S. Drakenberg T. Öhlin A.-K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Abstract Full Text PDF PubMed Google Scholar, 20Handford P.A. Baron M. Mayhew M. Wills A. Beesley T. Brownlee G.G. Campbell I.D. EMBO J. 1990; 9: 475-480Crossref PubMed Scopus (146) Google Scholar). The Ca2+affinity of isolated EGF-like modules is low and roughly equal (K d = 0.5–8 mm at 0.15 mNaCl) (19Persson E. Selander M. Linse S. Drakenberg T. Öhlin A.-K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Abstract Full Text PDF PubMed Google Scholar, 20Handford P.A. Baron M. Mayhew M. Wills A. Beesley T. Brownlee G.G. Campbell I.D. EMBO J. 1990; 9: 475-480Crossref PubMed Scopus (146) Google Scholar, 22Handford P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), 2Stenberg, Y., Julenius, K., Dahlqvist, I., Drakenberg, T., and Stenflo, J. (1997) Eur. J. Biochem., in press. 2Stenberg, Y., Julenius, K., Dahlqvist, I., Drakenberg, T., and Stenflo, J. (1997) Eur. J. Biochem., in press. although the reported Ca2+ affinities for EGF sites in intact proteins vary from moderate (K d = 0.1 mm) to very strong (K d ≤ 10 nm) (23Glanville R.W. Qian R.-Q. McClure D.W. Maslen C., L. J. Biol. Chem. 1994; 269: 26630-26634Abstract Full Text PDF PubMed Google Scholar, 24Dahlbäck B. Hildebrand B. Linse S. J. Biol. Chem. 1990; 265: 18481-18489Abstract Full Text PDF PubMed Google Scholar, 25Persson E. Björk I. Stenflo J. J. Biol. Chem. 1991; 266: 2444-2452Abstract Full Text PDF PubMed Google Scholar, 26Valcarce C. Selander-Sunnerhagen M. Tämlitz A.-M. Drakenberg T. Björk I. Stenflo J. J. Biol. Chem. 1993; 268: 26673-26678Abstract Full Text PDF PubMed Google Scholar, 27Knott V. Downing K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (98) Google Scholar). This is in accord with a large body of experimental data for different types of Ca2+-binding proteins. These data show that the observed Ca2+ affinity of a specific site is not governed solely by local interactions between the calcium ion and its coordinating oxygens provided by the backbone and side chains of the protein as well as water molecules. Major contributions are provided by other parts of the protein, and long range interactions can have profound effects on affinity (28Linse S. Forsén S. Adv. Second Messenger Phosphoprotein Res. 1995; 30: 89-151Crossref PubMed Scopus (139) Google Scholar, 29Falke J.J. Drake S.K. Hazard A.L. Pearsen O.B. Q. Rev. Biophys. 1994; 27: 219-290Crossref PubMed Scopus (336) Google Scholar). In coagulation factors IX and X, the adjacent Gla-containing module increases the affinity of the EGF site approximately 10-fold to aK d ≈ 1 × 10−4m, making the site essentially saturated at physiological Ca2+concentrations (26Valcarce C. Selander-Sunnerhagen M. Tämlitz A.-M. Drakenberg T. Björk I. Stenflo J. J. Biol. Chem. 1993; 268: 26673-26678Abstract Full Text PDF PubMed Google Scholar). We have found that the isolated third and fourth EGF modules from protein S bind Ca2+ with aK d of 5.2 mm and 0.6 mm, respectively.2 However, in intact protein S there appears to be four very high affinity Ca2+-binding sites (K d ≈ 10−9–10−7m), at least one of which appears to be located in the EGF module region (24Dahlbäck B. Hildebrand B. Linse S. J. Biol. Chem. 1990; 265: 18481-18489Abstract Full Text PDF PubMed Google Scholar).Fibrillin, a structural component of connective tissue microfibrils, contains 47 EGF modules, 43 of which have the Ca2+ binding motif (22Handford P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Glanville R.W. Qian R.-Q. McClure D.W. Maslen C., L. J. Biol. Chem. 1994; 269: 26630-26634Abstract Full Text PDF PubMed Google Scholar, 31Wu Y.-S. Bevilacqua L.H. Berg J.M. Curr. Biol. 1995; 2: 91-97Scopus (35) Google Scholar). In pairs of EGF modules from fibrillin, others have found the Ca2+ affinity of the C-terminal module to beK d ≈ 0.35 mm (22Handford P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 27Knott V. Downing K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (98) Google Scholar), which is approximately 25-fold higher than the Ca2+ affinity found in the isolated modules. Yet the N-terminal module appeared not to contribute additional ligands to the calcium ion in the C-terminal module (27Knott V. Downing K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (98) Google Scholar). Downing et al. (33Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) proposed the increased affinity to be attributable to shielding of the calcium ion from solvent and the provision of a more defined binding site by the interdomain interface.To shed light on the effect of an adjacent EGF module on the Ca2+ affinity of its neighbor, we have expressed fragments containing two, three, or four Ca2+ binding EGF modules from human protein S in Spodoptera cells using baculovirus. The expression and characterization of the fragments are described in an earlier report. 3Y. Stenberg, B. Dahlbäck, and J. Stenflo, submitted for publication. 3Y. Stenberg, B. Dahlbäck, and J. Stenflo, submitted for publication. We have now determined the Ca2+ affinity of the recombinant modules using a Ca2+ complexation method and 1H NMR spectroscopy. EGF modules 3 and 4 each contains one very high affinity Ca2+-binding site (≤K d of 10−10–10−8m), whereas there is no site of comparable affinity in EGF 1 or 2. Moreover, the Ca2+-binding site in EGF 3 is much stronger in the presence of EGF 4. Module-module interactions thus have a major effect on the Ca2+ affinity of these module oligomers and, presumably, even more so in the intact proteins. However, the nature of these interactions has yet to be eluciated in molecular detail.DISCUSSIONProtein S is the only vitamin K-dependent coagulation factor that is not a serine protease. Moreover, the N-terminal part of protein S has a modular structure that differs from that of factors VII, IX, and X and protein C. Whereas these proteins all have two EGF modules immediately C-terminal of the Gla module, protein S has a module with a thrombin-sensitive peptide bond between the Gla module and the four EGF modules. In factors VII, IX, and X and protein C, only the N-terminal EGF module has the Ca2+ binding and Asp/Asn-β-hydroxylation sequence motifs. In protein S, all four EGF modules have the hydroxylation motif with partial hydroxylation of Asp in the first module and of Asn in the three following ones. The first EGF module of protein S is atypical, however, in that it has the β-hydroxylation motif but lacks the characteristic N-terminal motif required for Ca2+ binding (Fig. 5).The present studies performed on recombinant human EGF modules from protein S demonstrated the presence of very high affinity Ca2+ binding sites in the EGF module region of the protein. The studies also demonstrated the high Ca2+ affinity to be independent of the Gla module. This is in good agreement with previous Ca2+ blotting experiments performed on bovine and human protein S (24Dahlbäck B. Hildebrand B. Linse S. J. Biol. Chem. 1990; 265: 18481-18489Abstract Full Text PDF PubMed Google Scholar).3 The affinity of one of the Ca2+ sites in EGF 1–4 and 2–4 was too high to allow an accurate estimate of the binding constant with the Quin 2 method,i.e. K d < 10−10m (in the absence of NaCl). This site, as well as a second high affinity site (K d ≈ 2–3 × 10−8min the absence of NaCl), appeared to be located in the third or fourth EGF module, as judged by the Ca2+ titrations. In the presence of 0.15 m NaCl, the Ca2+ affinity of the sites in EGF 1–4 and 2–4 decreased 10–1000-fold, and the affinity of the site in EGF 1–3 became too low to be measured accurately with 5,5′-Br2BAPTA. This is consistent with all the binding sites in highly charged environments. Similar salt effects have been found in studies of Ca2+-binding sites in calmodulin and calbindin D9K (34Linse S. Johansson C. Brodin P. Grundström T. Drakenberg T. Forsén S. Biochemistry. 1991; 30: 154-162Crossref PubMed Scopus (129) Google Scholar, 39Linse S. Helmersson A. Forsén S. J. Biol. Chem. 1991; 266: 8050-8054Abstract Full Text PDF PubMed Google Scholar). In contrast, the effect of salt on the Ca2+ affinity of the isolated EGF 3 (K d = 5.2 mm in the absence of salt and 6.1 mm in the presence of 0.15 m NaCl) was negligible and for EGF 4 (K d = 0.6 mm in the absence of salt and 8.6 mm in the presence of 0.15m NaCl) reduced the affinity by about 10-fold, as has previously been observed in the isolated EGF modules from factors IX and X (14Handford P.A. Mayhew M. Baron M. Winship P.R. Campbell I.D. Brownlee G.G. Nature. 1991; 351: 164-167Crossref PubMed Scopus (245) Google Scholar, 19Persson E. Selander M. Linse S. Drakenberg T. Öhlin A.-K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Abstract Full Text PDF PubMed Google Scholar).2 The Ca2+ affinity of EGF 3 and 4 in EGF 1–4 and EGF 2–4 in the presence of 0.15 m NaCl is 103- and 105-fold higher than in the corresponding synthetic isolated modules.2 The present data show that a major part of the increase in Ca2+ affinity for EGF 3 and EGF 4, when going from isolated module to intact protein, stems from interactions with a neighboring EGF module in the protein.A noteworthy result in this study was that EGF 3 has a Ca2+-binding site manifesting higher Ca2+affinity in fragment EGF 2–4 than it does in fragment EGF 1–3, (K d ≈ 2 × 10−8m versus K d ≈ 7 × 10−6m); i.e. EGF 3 appears to have an at least 350-fold lower Ca2+ affinity in EGF 1–3 than in EGF 2–4. It has been shown in several cases that the Ca2+ affinity of an EGF module is reduced if the protein segment N-terminal to it is removed. This may be an effect of stabilization of the site, since one or two of the Ca2+ ligands may be located N-terminal to the module. The data obtained for the site in EGF 3 demonstrates an influence on Ca2+ affinity from the module on the C-terminal site. However, we suspect that EGF 4 has higher affinity than EGF 3 because of extra negative charge in its N-terminal region (see below). If EGF 3 has the highest affinity site, then its affinity would have been increased by the addition of EGF 4. Also EGF 2 has ∼25-fold higher Ca2+ affinity in EGF 1–4 than in EGF 1–3; this is also influenced by EGF 4. EGF 1 seems to influence EGF 2 but to have no influence on EGF 3 or 4. To clarify the interactions between modules in detail, access to recombinant EGF 3–4 would have been helpful. Unfortunately, at this stage we could not investigate the Ca2+ binding constants further, as attempts to express a construct encompassing modules 3–4 were fraught with difficulty because the recombinant protein yielded only a smear and high molecular weight oligomers when analyzed by SDS-polyacrylamide gel electrophoresis (attempts to express EGF 1–2 were beset with similar problems).In this context, it should be borne in mind that EGF 4 in human protein S has the sequence EDIDE (positions 201–205; Fig. 5), whereas bovine protein S has the sequence DDVDE in the corresponding region,i.e. in both species, EGF 4 has an extra negative charge that is not found in most other Ca2+ binding EGF modules. This may contribute to the very high affinity of the Ca2+site but does not in itself suffice to explain the difference in Ca2+ affinity compared with for instance, fibrillin (K d ≈ 0.35 mm in the C-terminal module in a module pair) (27Knott V. Downing K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (98) Google Scholar). The Ca2+ binding properties of other EGF modules with the extra negative charge have yet to be reported.EGF modules 2, 3, and 4 from protein S are similar to most other Ca2+ binding EGF modules, e.g. from thrombomodulin and fibrillin, in that the sequence motif DI/VDE before the first Cys residue in the module is conserved. The other coagulation factors form a unique group in that they all have the sequence DGDQ before the first Cys residue in the N-terminal EGF module (7Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (328) Google Scholar, 12Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar). Moreover, they have erythro-β-hydroxyaspartic acid/Asp where EGF 2, 3, and 4 from protein S haveerythro-β-hydroxyasparagine/Asn. Yet, the Ca2+affinity of the isolated EGF modules from factors IX and X is in the same range as those from protein S and fibrillin, i.e. K d ≈ 1 mm (14Handford P.A. Mayhew M. Baron M. Winship P.R. Campbell I.D. Brownlee G.G. Nature. 1991; 351: 164-167Crossref PubMed Scopus (245) Google Scholar, 19Persson E. Selander M. Linse S. Drakenberg T. Öhlin A.-K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Abstract Full Text PDF PubMed Google Scholar, 30Selander-Sunnerhagen M. Persson E. Dahlqvist I. Drakenberg T. Stenflo J. Mayhew M. Robin M. Handford P. Tilley J.W. Campbell I.D. Brownlee G.G. J. Biol. Chem. 1993; 268: 23339-23344Abstract Full Text PDF PubMed Google Scholar). The function of the conserved Gly residue is not known. At this stage it can only be speculated that the adjacent C-terminal α-helix in the Gla module can not accomodate the bulky side chain of an Ile/Val residue in this position for steric reasons but requires a small residue such as glycine.The present and previous studies demonstrate that EGF modules can provide extremely versatile Ca2+-binding sites in extracellular and membrane proteins. The wide span of Ca2+affinities, from K d values in the nanomolar to the millimolar range, is striking. How the structure accounts for the differences in Ca2+ affinity, in addition to the apparent variations in net negative charge in the vicinity of the site, is not clear in the absence of a high resolution structure for protein S. The same Ca2+ ligands were identified in the determination of the structure of the Ca2+ form of the isolated EGF modules from factor X (NMR spectroscopy) and factor IX (x-ray crystallography) (21Selander-Sunnerhagen M. Ullner M. Persson E. Teleman O. Stenflo J. Drakenberg T. J. Biol. Chem. 1992; 267: 19642-19649Abstract Full Text PDF PubMed Google Scholar, 33Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The EGF module pair from fibrillin binds Ca2+with approximately 10-fold higher affinity than does the isolated modules from factors IX and X (27Knott V. Downing K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (98) Google Scholar, 30Selander-Sunnerhagen M. Persson E. Dahlqvist I. Drakenberg T. Stenflo J. Mayhew M. Robin M. Handford P. Tilley J.W. Campbell I.D. Brownlee G.G. J. Biol. Chem. 1993; 268: 23339-23344Abstract Full Text PDF PubMed Google Scholar). The x-ray structure of the module pair did not lead to the identification of additional Ca2+ ligands. Instead, Downing et al. proposed the increased Ca2+ affinity to be attributable to shielding of the calcium ion from the solvent and the provision of a better-defined binding site by the intermodular surface (33Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The present findings for protein S suggest that EGF 4 increases the Ca2+ affinities of the sites in EGF 3 and EGF 2 in a manner that remains to be elucidated. In this context, it is also of interest that EGF 1 is crucial for the inhibitory activity of EGF 1–4 on the interaction between activated protein C and protein S.3However, EGF 1–4 is 10-fold more active in this respect than EGF 1–3, suggesting extensive module-module interactions among all four EGF modules).3Determination of the solution structure of a pair of Ca2+binding EGF modules from fibrillin has shown them to be oriented in a near linear arrangement that is stabilized by Ca2+ligation. The orientation of the EGF modules in protein S is not known. Nor do we know the biological effects of the high variation in Ca2+ affinity between the EGF modules in protein S. Elucidation of the importance of Ca2+ binding in module-module interactions appears to be a prerequisite for an understanding of the function of these modules in coagulation factors as well as in the more complex receptors. Protein S is a vitamin K-dependent plasma protein that functions as a cofactor to activated protein C, a regulator of blood coagulation, by enhancing the activated protein C-mediated rates of degradation of factors Va and VIIIa (1Dahlbäck B. Thromb. Haemostasis. 1991; 66: 49-61Crossref PubMed Scopus (323) Google Scholar, 2Dahlbäck B. Stenflo J. Stamatoyannopoulos G. Nienhuis A.W. Majerus P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Co., Philadelphia1994: 599-628Google Scholar, 3Dahlbäck D. Stenflo J. Bloom A.L. Forbes C.D. Thomas D.P. Tuddenham E.G.D. Haemostasis and Thrombosis. 1. Churchill Livingstone, Edinburgh, UK1994: 671-698Google Scholar). Protein S consists of an N-terminal γ-carboxyglutamic acid (Gla) 1The abbreviations used are: Gla, γ-carboxyglutamic acid; 5,5′-Br2BAPTA, 5,5′-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; EGF, epidermal growth factor; EGF 1–3, residues 73–203; EGF 1–4, residues 73–245; EGF 2–3, residues 115–203; and EGF 2–4, residues 115–245, all in human protein S; SF 9 cells, Spodoptera frugiperda ovarian cells. 1The abbreviations used are: Gla, γ-carboxyglutamic acid; 5,5′-Br2BAPTA, 5,5′-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; EGF, epidermal growth factor; EGF 1–3, residues 73–203; EGF 1–4, residues 73–245; EGF 2–3, residues 115–203; and EGF 2–4, residues 115–245, all in human protein S; SF 9 cells, Spodoptera frugiperda ovarian cells.-containing module that is followed by a module with a thrombin-sensitive peptide bond and four epidermal growth factor (EGF)-like modules, whereas the C-terminal half of the molecule is occupied by a module that is homologous to steroid hormone-binding proteins (4Dahlbäck B. Lundwall Å. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4199-4203Crossref PubMed Scopus (106) Google Scholar, 5Lundwall Å. Dackowski W. Cohen E.H. Shaffer M. Mahr A. Dahlbäck B. Stenflo J. Wydro R.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Crossref PubMed Scopus (149) Google Scholar, 6Gershagen S. Fernlund P. Lundwall Å. FEBS Lett. 1987; 220: 129-135Crossref PubMed Scopus (77) Google Scholar, 7Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (328) Google Scholar). Calcium binding to vitamin K-dependent clotting factors, including protein S, is complex, >10 calcium ions being bound to 2 or 3 types of sites that profoundly influence the function of these proteins (8Stenflo J. Dahlbäck B. Stamatoyannopoulos G. Nienhuis A.W. Majerus P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Co., Philadelphia1994: 565-598Google Scholar). The Gla module binds Ca2+ and endows protein S with membrane affinity, whereas the EGF module-containing part of protein S appears to be involved in the interaction with activated protein C (9Dahlbäck B. Hildebrand B. Malm J. J. Biol. Chem. 1990; 265: 8127-8135Abstract Full Text PDF PubMed Google Scholar,10He X. Shen L. Dahlbäck B. Eur. J. Biochem. 1995; 227: 433-440Crossref PubMed Scopus (39) Google Scholar). The EGF modules in protein S are members of a large family of similar modules that are found in extracellular and membrane mosaic proteins in species ranging from Caenorhabditis elegans toDrosophila melanogaster and humans (7Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (328) Google Scholar, 11Appella E. Weber I.T. Blasi F. FEBS Lett. 1988; 231: 1-4Crossref PubMed Scopus (236) Google Scholar, 12Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar). Several of these proteins are involved in cell differentiation, blood coagulation and fibrinolysis, and in
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