Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs.
Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.
Spratt
Donald E
DE
Department of Chemistry, University of Waterloo, ON, Canada.
Newman
Elena
E
Mosher
Jennifer
J
Ghosh
Dipak K
DK
Salerno
John C
JC
Guillemette
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D020832:EF Hand Motifs; D004579:Electron Transport; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007527:Isoenzymes; D008969:Molecular Sequence Data; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D007527:Isoenzymes; D010108:Oxyhemoglobins; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; D019255:NADPH Oxidases