pH-Dependent Structural Changes at Ca²⁺-binding Sites of Coagulation Factor IX-binding Protein

详细信息	查看全文 | 推荐本文 |

• 作者：Nobuhiro Suzuki ; Zui Fujimoto ; Takashi Morita ; Akiyoshi Fukamizu and Hiroshi Mizuno
• 刊名：Journal of Molecular Biology
• 出版年：2005
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：2005
• 卷：353
• 期：1
• 页码：80-87
• 文件大小：543 K

摘要

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 Ca²⁺-binding site with a unique affinity (K_d values of 14 μM and 130 μM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca²⁺ binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca²⁺ release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca²⁺-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 Ca²⁺-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 Ca²⁺ 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 pH 6.5, which may correspond to the step of Ca²⁺ release.