The C₂B domain of synaptotagmin I is a Ca²⁺-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C₂ domains and acts as a Ca²⁺ sensor in neurotransmitter release. The Ca²⁺-binding properties of the synaptotagmin I C₂A domain have been well characterized, but those of the C₂B domain are unclear. The C₂B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca²⁺-dependent manner, leading to an attractive model whereby Ca²⁺-dependent multimerization of synaptotagmin I via the C₂B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca²⁺-dependent C₂B domain dimerization, as well as in analyses of other C₂B domain interactions. To shed light on these issues, the C₂B domain has now been studied using biophysical techniques. The recombinant C₂B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C₂B domain interactions, including Ca²⁺-dependent dimerization. NMR experiments show that the pure recombinant C₂B domain binds Ca²⁺ directly but does not dimerize upon Ca²⁺ binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C₂A and C₂B domains, participates in a high molecular weight complex as a function of Ca²⁺. These results show that the recombinant C₂B domain of synaptotagmin I is a monomeric, autonomously folded Ca²⁺-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C₂B domains or requires a posttranslational modification.