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 aCa(2+) sensor in neurotransmitter release. The Ca2+-binding pr operties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C2B domain are unclear. The C2B domain was previously foun d to pull down synaptotagmin I from brain homogenates in a Ca2+-dependent m anner, lending to an attractive model whereby Ca2+-dependent multimerizatio n of synaptotagmin I via the C2B domain participates in fusion pore formati on. However, contradictory results have been described in studies of Ca2+-d ependent C2B domain dimerization, as well as in analyses of other C2B domai n 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 ti ghtly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously impl icated in several C2B domain interactions, including Ca2+-dependent dimeriz ation. NMR experiments show that the pure recombinant C2B domain binds Ca2 directly but does not dimerize upon Ca2+ binding. In contrast, a cytoplasm ic fragment of native synaptotagmin I from brain homogenates, which include s 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 o f synaptotagmin I is a monomeric, autonomously folded Ca2+-binding,(C) modu le 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.