title: Structural and mutational analysis of functional differentiation between synaptotagmins-1 and -7.
abstract: Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ 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 C2B domain replaced by the synaptotagmin-7 C2B 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 C2B domain, we determined its crystal structure at 1.44 A resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B 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 C2B 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.
authors.affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Mingshan
authors.initials: M
authors.lastname: Xue
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Craig
authors.forename: Ok-Ho
authors.initials: OH
authors.lastname: Shin
authors.forename: Liyi
authors.initials: L
authors.lastname: Li
authors.forename: Chad A
authors.initials: CA
authors.lastname: Brautigam
authors.forename: Diana R
authors.initials: DR
authors.lastname: Tomchick
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
authors.forename: Christian
authors.initials: C
authors.lastname: Rosenmund
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D050863:Synaptotagmin I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D050857:Synaptotagmins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D018377:Neurotransmitter Agents; D050863:Synaptotagmin I; D050857:Synaptotagmins; D002118:Calcium
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