N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor

Neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca2+ binding and Ca2+-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The Ca-45 binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca2+ more strongly ( with three Ca2+ binding sites) than the non-myristoylated one ( with two Ca2+ binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca2+. Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca2+ binding were not seen in E120Q mutant ( disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca2+ also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg2+-induced changes in near-UV CD are different from Ca2+-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca2+, which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca2+ binding, which might be crucial for its physiological functions.