NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1) |
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Authors: | Karuppanan Muthusamy Kathir Li Gao Dakshinamurthy Rajalingam Sherri Brixey Dan Davis Igor Prudovsky |
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Institution: | a Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA b Maine Medical Center Research Institute, Scarborough, ME 04074, USA |
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Abstract: | Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu2+) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu2+ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu2+ appears to stabilize the protein bound to pS vesicles. Cu2+ and lipid binding interface mapped using 2D 1H-15N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu2+ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu2+, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu2+-induced non-classical secretion of hFGF-1. |
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Keywords: | hFGF-1 human fibroblast growth factor-1 Syt1 Synaptotagmin 1 HSQC Heteronuclear Single Quantum Coherence NMR nuclear magnetic resonance |
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