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)

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.

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

“…Even though line broadening could limit the use of NMR spectra, it is a helpful approach to study metal‐binding sites and to investigate protein active sites. Indeed, line broadening due to interactions with paramagnetic metal ions has been used as an effective way to probe copper‐binding sites in small molecule and macromolecules . Paramagnetic NMR spectroscopy has also been used to determine copper‐binding sites and to study copper coordination with prion proteins …”

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor

“…Bacterial expression and purification of the C2B domain of Syt1 were carried out as described in detail [45]. Briefly, cDNA encoding the C2B domain of synaptotagmin I (residues 270 to 421) and p40 Syt1 was kindly provided by Professor Thomas Sudhof.…”

NMR of liquid crystals and micellar solutions

“…Cu 2 þ and lipid binding interface mapped using 2D 1 H-15 N heteronuclear single quantum coherence experiments reveal that residues in b-strand I contributes to the unique Cu 2 þ binding site in the C2B domain. 48 Phospholamban (PLB) is an integral membrane protein that regulates Ca 2 þ transport through an inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca 2 þ and of cardiac contractility in vivo.…”