title: The C2A domain of synaptotagmin exhibits a high binding affinity for copper: implications in the formation of the multiprotein FGF release complex.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
authors.forename: Dakshinamurthy
authors.initials: D
authors.lastname: Rajalingam
authors.forename: Thallapuranam Krishnaswamy S
authors.initials: TK
authors.lastname: Kumar
authors.forename: Chin
authors.initials: C
authors.lastname: Yu
mesh_terms: D000818:Animals; D003300:Copper / Q000378:metabolism*; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D046912:Multiprotein Complexes; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D050863:Synaptotagmin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D016220:Fibroblast Growth Factor 1; D003300:Copper