NMR STUDIES OF C2 DOMAINS OF SYNAPTOTAGMIN I

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The synaptotagmin is involved in the Ca2+-induced neurotrasmitter release. The synaptotagmin has roughly 4 domains: intravesicular domain, transmembrane domain, and 2 cytosolic domains. Two cytosolic domains are named C2A and C2B. The first domain is the one to study by NMR. C2A domain is known to be a Ca2+ sensor in the membrane exocytosis. According to the Ca2+ binding assay in the presence of phospholipids, the Hill coefficient is larger than 3, which in turn means C2A should bind at least 3 Ca2+ ions. So far, the x-ray structure found one Ca2+ binding site, and NMR data suggested the second site. Very recently, the x-ray structure of the PLC-?1 which has a C2 domain proposed the third binding site based on the homology of the two structures. The failure in finding the third binding site is presumed to result from the absence of the lipids since the intrinsic affinity of C2A domain to Ca2+ is low. In the current NMR study of this C2A domain, a short version of the phospholipid has been used. NMR data show changes in the chemical shifts with the presence of Ca2+ or lipid or both, which strongly suggests a existence of a ternary complex. The goal of this study is to solve the structure of the ternary complex: C2A-Ca2+-lipid.