The C2A Domain of Synaptotagmin Exhibits a High Binding Affinity for Copper: Implications in the Formation of the Multiprotein FGF Release Complex

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. Cu2+ is known to play an important role in the formation of the multiprotein release complex. The source of Cu2+ 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 Cu2+ ions with high affinity. Results from the isothermal calorimetry (ITC), near-UV circular dichroism (CD), and absorption spectroscopy experiments suggest that four Cu2+ 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 Cu2+. Competition experiments monitored by ITC and fluorescence resonance energy transfer indicate that Cu2+ and Ca2+ ions share common binding sites on the C2A domain. Cu2+ ions compete with and replace Ca2+ ions bound to the C2A domain. Two-dimensional nuclear magnetic resonance spectroscopy data clearly show that Cu2+ ions bind to the Ca2+ 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 Cu2+ binding site located in the loop connecting β-strands 7 and 8. It appears that the C2A domain provides the Cu2+ ions required for the formation of the multiprotein FGF release complex.