title: Role of electrostatic and hydrophobic interactions in Ca(2+)-dependent phospholipid binding by the C(2)A-domain from synaptotagmin I.
abstract: Most C(2)-domains bind to phospholipid bilayers as a function of Ca(2+). Although phospholipid binding is central for the normal functions of C(2)-domain proteins, the precise mechanism of phospholipid binding is unclear. One of the key questions is whether phospholipid binding by C(2)-domains is primarily governed by electrostatic or hydrophobic interactions. We have now examined this question for the C(2)A-domain of synaptotagmin I, a membrane protein of secretory vesicles with an essential function in Ca(2+)-triggered exocytosis. Our results confirm previous data showing that Ca(2+)-dependent phospholipid binding by the synaptotagmin C(2)A-domain is exquisitely sensitive to ionic strength, suggesting an essential role for electrostatic interactions. However, we find that hydrophobic interactions mediated by exposed residues in the Ca(2+)-binding loops of the C(2)A-domain, in particular methionine 173, are also essential for tight phospholipid binding. Furthermore, we demonstrate that the apparent Ca(2+) affinity of the C(2)A-domain is determined not only by electrostatic interactions as shown previously, but also by hydrophobic interactions. Together these data indicate that phospholipid binding by the C(2)A-domain, although triggered by an electrostatic Ca(2+)-dependent switch, is stabilized by a hydrophobic mechanism. As a result, Ca(2+)-dependent phospholipid binding proceeds by a multimodal mechanism that mirrors the amphipathic nature of the phospholipid bilayer. The complex phospholipid binding mode of synaptotagmins may be important for its role in regulated exocytosis of secretory granules and synaptic vesicles.
authors.affiliation: Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, Dallas, Texas, USA.
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Gerber
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D004563:Electrochemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D050863:Synaptotagmin I; D050857:Synaptotagmins; D002118:Calcium