Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

Abstract

C 2 domains are the second most widespread Ca 2+ -binding modules present in nature according to analyses of the human genome 1 and Ca 2+ -dependent phospholipid binding constitutes their most general activity 2 . The sequence determinants of this activity have been particularly well characterized for the two C 2 domains that form most of the cytoplasmic region of synaptotagmin 1 (referred to as the C 2 A and C 2 B domains). This synaptic vesicle protein functions in triggering fast neurotransmitter release by binding Ca 2+ through both of its C 2 domains 3,4 , and is the most conserved among the synaptotagmins, a family of membrane-trafficking proteins with at least 8 isoforms in D. melanogaster and 15 in mammals Biochemical analyses of additional synaptotagmins have suggested that they form a hierarchy of Ca 2+ sensors with different Ca 2+ affinities RESULTS Intrinsic Ca 2+ binding to synaptotagmins 4 and 11 To analyze the intrinsic Ca 2+ -binding properties of the rat and D. melanogaster synaptotagmin 4 C 2 domains, we used 1 H-15 N heteronuclear single quantum correlation (HSQC) spectra. Ca 2+ induced The neuronal protein synaptotagmin 1 functions as a Ca 2+ sensor in exocytosis via two Ca 2+ -binding C 2 domains. The very similar synaptotagmin 4, which includes all the predicted Ca 2+ -binding residues in the C 2 B domain but not in the C 2 A domain, is also thought to function as a neuronal Ca 2+ sensor. Here we show that, unexpectedly, both C 2 domains of fly synaptotagmin 4 exhibit Ca 2+ -dependent phospholipid binding, whereas neither C 2 domain of rat synaptotagmin 4 binds Ca 2+ or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca 2+ ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C 2 B domain unable to form full Ca 2+ -binding sites. These results indicate that synaptotagmin 4 is a Ca 2+ sensor in the fly but not in the rat, that the Ca 2+ -binding properties of C 2 domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties