title: Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor.
abstract: Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released through a Cu(2+)-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu(2+)-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu(2+) to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu(2+) and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu(2+) in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb(3+) show that there are two Cu(2+)-binding pockets on the C2B domain, and one of these is also a Ca(2+)-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu(2+). The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: sjayanth@uark.edu.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Jayanthi
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: kmkathir@gmail.com.
authors.forename: Karuppanan Muthusamy
authors.initials: KM
authors.lastname: Kathir
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: rajalingam.dakshinamurthy@wku.edu.
authors.forename: Dakshinamurthy
authors.initials: D
authors.lastname: Rajalingam
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: mfurr@email.uark.edu.
authors.forename: Mercede
authors.initials: M
authors.lastname: Furr
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: dailyphd@gmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Daily
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: rdt001@uark.edu.
authors.forename: Ryan
authors.initials: R
authors.lastname: Thurman
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: lnrutherford@gmail.com.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Rutherford
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: rxc018@uark.edu.
authors.forename: Reena
authors.initials: R
authors.lastname: Chandrashekar
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: pxa001@uark.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Adams
authors.affiliation: Maine Medical Center Research Institute, Scarborough, ME 04074, USA. Electronic address: prudoi@mmc.org.
authors.forename: Igor
authors.initials: I
authors.lastname: Prudovsky
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: sthalla@uark.edu.
authors.forename: Thallapuranam Krishnaswamy Suresh
authors.initials: TK
authors.lastname: Kumar
keywords: Fibroblast growth factor; Lipid binding; Nonclassical; Secretion; Signal peptide; Synaptotagmin