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

Synaptotagmin I is a synaptic vesicle protein that contains two C 2 domains and acts as a Ca 2+ sensor in neurotransmitter release. The Ca 2+ -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 Ca 2+ -dependent manner, leading to an attractive model whereby Ca 2+ -dependent multimerization of synaptotagmin I via the C2B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca 2+ -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 Ca 2+ -dependent dimerization. NMR experiments show that the pure recombinant C2B domain binds Ca 2+ directly but does not dimerize upon Ca 2+ 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 Ca 2+ . These results show that the recombinant C2B domain of synaptotagmin I is a monomeric, autonomously folded Ca 2+ -binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C 2B domains or requires a posttranslational modification.