RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1α C 2B domain. We also find that the isolated RIM1α C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1α C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1α C 2B domain forms weak dimers in solution. The crystal structure of the RIM1α C2B domain dimer at 1.7 Å resolution reveals that it forms a β-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the β-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.
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