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 C₂ domains. The very similar synaptotagmin 4, which includes all the predicted Ca²⁺- binding residues in the C₂B domain but not in the C₂A domain, is also thought to function as a neuronal Ca²⁺ sensor. Here we show that, unexpectedly, both C₂ 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 C ²⁺-binding properties of C₂ domains cannot be reliab y predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.