Structural and Mutational Analysis of Functional Differentiation between Synaptotagmins-1 and -7

Synaptotagmins are known to mediate diverse forms of Ca(2+)-triggered exocytosis through their C(2) domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca(2+) sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca(2+) sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C(2)B domain replaced by the synaptotagmin-7 C(2)B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C(2)B domain, we determined its crystal structure at 1.44 Å resolution. The synaptotagmin-7 C(2)B domain structure is very similar to that of the synaptotagmin-1 C(2)B domain and contains three Ca(2+)-binding sites. Two of the Ca(2+)-binding sites of the synaptotagmin-7 C(2)B domain are also present in the synaptotagmin-1 C(2)B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C(2)B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.