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

The neuronal protein synaptotagmin 1 functions as a Ca2+ sensor in exocytosis via two Ca2+-binding C-2 domains. The very similar synaptotagmin 4, which includes all the predicted Ca2+-binding residues in the C2B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca2+ sensor. Here we show that, unexpectedly, both C-2 domains of fly synaptotagmin 4 exhibit Ca2+-dependent phospholipid binding, whereas neither C-2 domain of rat synaptotagmin 4 binds Ca2+ or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca2+ ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. These results indicate that synaptotagmin 4 is a Ca2+ sensor in the fly but not in the rat, that the Ca2+-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.