Abstract
Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-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 Ca(2+) binding and Ca(2+)-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The (45)Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca(2+) more strongly (with three Ca(2+) binding sites) than the non-myristoylated one (with two Ca(2+) binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca(2+). 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 Ca(2+) 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 Ca(2+) also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg(2+)-induced changes in near-UV CD are different from Ca(2+)-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca(2+), which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca(2+) binding, which might be crucial for its physiological functions.
Highlights
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 has been known that the Ca2ϩ-myristoyl switch plays a significant role in the Ca2ϩ sensor family of Ca2ϩ-binding proteins where, upon Ca2ϩ binding to EF-hands, the generally buried N-terminal myristoyl chain protrudes out, facilitating membrane and target binding [12, 35]
We have seen that Ca2ϩ binding to myristoylated protein induces comparatively large conformational changes (Table III)
Summary
Vol 279, No 26, Issue of June 25, pp. 27158 –27167, 2004 Printed in U.S.A. N-terminal Myristoylation Regulates Calcium-induced Conformational Changes in Neuronal Calcium Sensor-1*. Of all of the aforementioned NCS proteins, recoverin is the most studied protein It is a Ca2ϩ sensor in retinal rod and cone cells wherein, besides the EF-1, EF-4 is non-functional and Ca2ϩ binds only to EF-2 and EF-3 sites, leading to the exposure of the buried N-terminal myristoyl group known as Ca2ϩ-myristoyl switch [12]. The data for affinity of mammalianmyristoylated NCS-1 (the biologically active form in a variety of assays) are not available and conflicting results exist for non-myristoylated NCS-1 [24] and yeast Frq1 [20] In view of these discrepancies, the exact role of myristoylation on the structure and function of NCS-1 is not yet clear. We observe that myristoylation regulates and restricts the Ca2ϩ binding affinity and conformational changes by increasing the cooperativity for ion binding This is of physiological significance, because Ca2ϩ binding renders these proteins active for their functional roles by introducing conformational changes. The Ca2ϩ-myristoyl switch is functional in the E120Q mutant, other properties such as Ca2ϩ-dependent surface hydrophobicity were altered by this mutation
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