title: The calcium-dependent and calcium-independent membrane binding of synaptotagmin 1: two modes of C2B binding.
abstract: The Ca2+-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca2+-independent manner with a lipid partition coefficient of approximately 3.0 x 10(2) M(-1). A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca2+. Although the Ca2+-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca2+. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca2+, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca2+-binding loops of C2B insert into the bilayer. This Ca2+-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.
authors.affiliation: Department of Chemistry, McCormick Road, University of Virginia, Charlottesville, VA 22904-4319, USA.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Kuo
authors.forename: Dawn Z
authors.initials: DZ
authors.lastname: Herrick
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Ellena
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008567:Membranes, Artificial; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D050863:Synaptotagmin I / Q000737:chemistry* / Q000378:metabolism*; D014867:Water
chemical_list: D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D050863:Synaptotagmin I; D014867:Water; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine