N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1.
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.
Jeromin
Andreas
A
Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
Muralidhar
Dasari
D
Parameswaran
Malavika Nair
MN
Roder
John
J
Fairwell
Thomas
T
Scarlata
Suzanne
S
Dowal
Louisa
L
Mustafi
Sourajit M
SM
Chary
Kandala V R
KV
Sharma
Yogendra
Y
D019943:Amino Acid Substitution; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism*; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D008081:Liposomes; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C101464:frequenin calcium sensor proteins; D019814:Myristic Acid; D008274:Magnesium; D002118:Calcium