Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin.
Biochemical and genetic studies indicate that synaptotagmin I functions as a Ca2+ sensor during synaptic vesicle exocytosis and as a membrane receptor for the clathrin adaptor complex, AP-2, during endocytosis. These functions involve the interaction of two conserved domains, C2A and C2B, with effector proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and IV, and all three synaptotagmin isoforms bound the synprint region of the alpha1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding. Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two beta-strands. These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.
Chapman
E R
ER
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
Desai
R C
RC
Davis
A F
AF
Tornehl
C K
CK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001704:Biopolymers; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017386:Sequence Homology, Amino Acid; D050863:Synaptotagmin I; D050857:Synaptotagmins; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*
D001704:Biopolymers; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D050857:Synaptotagmins