title: pH-Dependent structural changes at Ca(2+)-binding sites of coagulation factor IX-binding protein.
abstract: Coagulation factor IX-binding protein, isolated from Trimeresurus flavoviridis (IX-bp), is a C-type lectin-like protein. It is an anticoagulant consisting of homologous subunits, A and B. Each subunit has a Ca(2+)-binding site with a unique affinity (K(d) values of 14muM and 130muM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca(2+) binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca(2+) release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca(2+)-binding sites with each other and with those of the solved structures under alkaline conditions; pH 7.8 and pH 8.0 (complexed form). At pH 6.5, Glu43 in the Ca(2+)-binding site of subunit A displayed two conformations. One (minor) is that in the alkaline state, and the other (major) is that at pH 4.6. However, the corresponding Gln43 residue of subunit B is in only a single conformation, which is almost identical with that in the alkaline state. At pH 4.6, Glu43 of subunit A adopts a conformation similar to that of the major conformer observed at pH 6.5, while Gln43 of subunit B assumes a new conformation, and both Ca(2+) positions are occupied by water molecules. These results showed that Glu43 of subunit A is much more sensitive to protonation than Gln43 of subunit B, and the conformational change of Glu43 occurs around pH6.5, which may correspond to the step of Ca(2+) release.
authors.affiliation: Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Suzuki
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Morita
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Fukamizu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mizuno
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins; D018360:Crystallography, X-Ray; D005164:Factor IX / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017841:Trimeresurus
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D021122:Protein Subunits; D005164:Factor IX; D002118:Calcium