Rajalingam Dakshinamurthy, Kumar Thallapuranam Krishnaswamy S, Yu Chin
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Biochemistry. 2005 Nov 8;44(44):14431-42. doi: 10.1021/bi051387r.
Human acidic fibroblast growth factor (hFGF-1) is a potent mitogen and is involved in the regulation of key cellular process such as angiogenesis, differentiation, and morphogenesis. hFGF-1 is a signal peptide-less protein that is released into the extracellular compartment as a multiprotein complex consisting of S100A13, synaptotagmin (Syt1), and a hFGF-1 homodimer. Cu(2+) is known to play an important role in the formation of the multiprotein release complex. The source of Cu(2+) required for the formation of the multiprotein release complex is not clear. In this study, we show that the cytoplasmic C2A domain of synaptotagmin binds to Cu(2+) ions with high affinity. Results from the isothermal calorimetry (ITC), near-UV circular dichroism (CD), and absorption spectroscopy experiments suggest that four Cu(2+) ions bind per molecule of C2A domain. Far-UV CD and limited trypsin digestion analysis reveal that the C2A domain undergoes a mild conformational change upon binding to Cu(2+). Competition experiments monitored by ITC and fluorescence resonance energy transfer indicate that Cu(2+) and Ca(2+) ions share common binding sites on the C2A domain. Cu(2+) ions compete with and replace Ca(2+) ions bound to the C2A domain. Two-dimensional nuclear magnetic resonance spectroscopy data clearly show that Cu(2+) ions bind to the Ca(2+) binding sites in the loops (loops 1-3) located at the apex of the structure of the C2A domain. In addition, there is a unique Cu(2+) binding site located in the loop connecting beta-strands 7 and 8. It appears that the C2A domain provides the Cu(2+) ions required for the formation of the multiprotein FGF release complex.
人酸性成纤维细胞生长因子（hFGF-1）是一种强效促有丝分裂原，参与血管生成、分化和形态发生等关键细胞过程的调控。hFGF-1是一种无信号肽的蛋白质，作为由S100A13、突触结合蛋白（Syt1）和hFGF-1同二聚体组成的多蛋白复合物释放到细胞外区室。已知Cu(2+)在多蛋白释放复合物的形成中起重要作用。多蛋白释放复合物形成所需的Cu(2+)来源尚不清楚。在本研究中，我们表明突触结合蛋白的胞质C2A结构域与Cu(2+)离子具有高亲和力结合。等温滴定量热法（ITC）、近紫外圆二色性（CD）和吸收光谱实验结果表明，每分子C2A结构域结合四个Cu(2+)离子。远紫外CD和有限胰蛋白酶消化分析表明，C2A结构域在与Cu(2+)结合后发生轻微构象变化。ITC和荧光共振能量转移监测的竞争实验表明，Cu(2+)和Ca(2+)离子在C2A结构域上共享共同的结合位点。Cu(2+)离子与结合在C2A结构域上的Ca(2+)离子竞争并取代它们。二维核磁共振光谱数据清楚地表明，Cu(2+)离子结合到位于C2A结构域结构顶端的环（环1-3）中的Ca(2+)结合位点。此外，在连接β链7和8的环中存在一个独特 的Cu(2+)结合位点。似乎C2A结构域提供了多蛋白FGF释放复合物形成所需的Cu(2+)离子。
