Characterization of the calmodulin-binding domain of rat cerebellar nitric oxide synthase.
Nitric oxide (NO) has recently been identified as an intercellular messenger which is involved in the regulation of neurotransmission, vasorelaxation, and cytotoxicity. In cerebellum and endothelium this compound is synthesized by "constitutive" nitric oxide synthases (NOS); these are Ca(2+)-calmodulin (CaM)-dependent enzymes. A potential CaM-binding domain for the CaM-dependent NOS has previously been identified in the gene sequence. In this work, a synthetic 23 residue peptide encompassing the putative CaM-binding domain of rat cerebellar NOS was studied. The constitutive NOS peptide binds to CaM in a calcium-dependent manner with 1:1 stoichiometry as determined by polyacrylamide gel electrophoresis of the peptide-CaM complex in 4 M urea. Circular dichroism studies showed that the peptide binds to CaM in an alpha-helical conformation. Binding of the constitutive NOS peptide inhibits the stimulatory effect of CaM on cyclic nucleotide phosphodiesterase. From competition experiments between the peptide and phosphodiesterase we have determined a Kd of 2.2 nM for the peptide-CaM complex. Two-dimensional NMR and circular dichroism studies were used to determine the structure of the peptide in aqueous solution. In addition, the effect of increasing amounts of trifluoroethanol on the peptide structure was investigated. It was found that the peptide can adopt an alpha-helical structure which bears close resemblance to the structure of the CaM-bound form of the CaM-binding domains of myosin light chain kinases.
Zhang
M
M
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Vogel
H J
HJ
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002531:Cerebellum / Q000201:enzymology; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases