The evolutionary pressure to inactivate: A subclass of synaptotagmins with an amino acid substitution that abolishes Ca²⁺ binding

Synaptotagmin I is a Ca²⁺-binding protein of synaptic vesicles that serves as a Ca²⁺ sensor for neurotransmitter release and was the first member found of a large family of trafficking proteins. We have now identified a novel synaptotagmin, synaptotagmin XI, that is highly expressed in brain and at lower levels in other tissues. Like other synaptotagmins, synaptotagmin XI has a single transmembrane region and two cytoplasmic Ca- domains but is most closely related to synaptotagmin IV with which it forms a new subclass of synaptotagmins. The first C₂-domain of synaptotagmin I (the C₂A-domain) binds phospholipids as a function of Ca²⁺ and contains a Ca²⁺-binding site, the C₂-motif, that binds at least two Ca²⁺ ions via five aspartate residues and is conserved in most C₂-domains (Shao, X., Davletov, B., Sutton, B., Sudhof, T. C., Rizo, J. R. (1996) Science 273, 248- 253). In the C₂A-domains of synaptotagmins IV and XI, however, one of the five Ca²⁺-binding aspartates in the C₂-motif is substituted for a serine, suggesting that these C₂-domains do not bind Ca²⁺. To test this, we produced recombinant C₂A-domains from synaptotagmins IV and XI with either wild type serine or mutant aspartate in the C₂-motif. Circular dichroism showed that Ca²⁺ stabilizes both mutant but not wild type C₂-domains against temperature-induced denaturation, indicating that the mutations restore Ca²⁺-binding to the wild type C₂-domains. Furthermore, wild type C₂A-domains of synaptotagmins IV and XI exhibited no Ca²⁺-dependent phospholipid binding, whereas mutant C₂A-domains bound phospholipids as a function of Ca²⁺ similarly to wild type synaptotagmin I. These experiments suggest that a class of synaptotagmins was selected during evolution in which the Ca²⁺-binding site of the C₂A-domain was inactivated by a single point mutation. Thus, synaptotagmins must have Ca²⁺-independent functions as well as Ca²⁺-dependent functions that are selectively maintained in distinct members of this gene family.