Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

C2 domains are widespread motifs that often serve as Ca 2+ -binding modules; some proteins have more than one copy. An open issue is whether these domains, when du- plicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca 2+ sensor for neuronal exocytosis. Substitu- tion of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca 2+ , and shifted the Ca 2+ depen- dency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions be- tween the tandem C2 domains of syt-1 contribute to excitation-secretion coupling.