Direct interaction of a Ca2+-binding loop of synaptotagmin with lipid bilayers.
Synaptotagmin 1 binds Ca2+ and membranes via its C2A-domain and plays an essential role in excitation-secretion coupling. In this study, we sought to identify Ca2+- and membrane-induced local conformational changes in the C2A-domain of synaptotagmin and to delineate the C2A-lipid binding interface. To address these questions native phenylalanine residues were replaced, at each face of the domain, with tryptophan reporters. Changes in tryptophanyl fluorescence indicated that Ca2+ induced long range conformational changes throughout C2A, including regions distant from an established Ca2+-binding site. Addition of liposomes resulted in Ca2+-dependent increases in the fluorescence of tryptophans 193, 231, and 234. Only the tryptophan residues at positions 234 and 231, which lie within a Ca2+-binding loop of C2A, exhibited liposome-induced blue shifts in their emission spectra. Quenching experiments, using membrane-imbedded doxyl spin labels, revealed that tryptophan residues 231 and 234 penetrated lipid bilayers. These data delineate the lipid binding interface of C2A and provide the first evidence for adjacent Ca2+- and lipid-binding sites within a C2-domain. The penetration of C2A into membranes may function to bring components of the fusion machinery into contact with the lipid bilayer to initiate exocytosis.
Chapman
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ER
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA. erchapma@facstaff.wisc.edu
Davis
A F
AF
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D008051:Lipid Bilayers; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium