Role of electrostatic and hydrophobic interactions in Ca²⁺-dependent phospholipid binding by the C₂A-domain from synaptotagmin I

Most C₂-domains bind to phospholipid bilayers as a function of Ca²⁺. Although phospholipid binding is central for the normal functions of C₂-domain proteins, the precise mechanism of phospholipid binding is unclear. One of the key questions is whether phospholipid binding by C₂-domains is primarily governed by electrostatic or hydrophobic interactions. We have now examined this question for the C₂A-domain of synaptotagmin I, a membrane protein of secretory vesicles with an essential function in Ca²⁺-triggered exocytosis. Our results confirm previous data showing that Ca²⁺-dependent phospholipid binding by the synaptotagmin C₂A-domain is exquisitely sensitive to ionic strength, suggesting an essential role for electrostatic interactions. However, we find that hydrophobic interactions mediated by exposed residues in the Ca²⁺-binding loops of the C₂A-domain, in particular methionine 173, are also essential for tight phospholipid binding. Furthermore, we demonstrate that the apparent Ca²⁺ affinity of the C₂A-domain is determined not only by electrostatic interactions as shown previously, but also by hydrophobic interactions. Together these data indicate that phospholipid binding by the C₂A-domain, although triggered by an electrostatic Ca^2+-dependent switch, is stabilized by a hydrophobic mechanism. As a result, Ca²⁺-dependent phospholipid binding proceeds by a multimodal mechanism that mirrors the amphipathic nature of the phospholipid bilayer. The complex phospholipid binding mode of synaptotagmins may be important for its role in regulated exocytosis of secretory granules and synaptic vesicles.