The calmodulin-nitric oxide synthase interaction. Critical role of the calmodulin latch domain in enzyme activation.
The neuronal isoform of nitric oxide synthase (nNOS) requires calmodulin for nitric oxide producing activity. Calmodulin functions as a molecular switch, allowing electron transport from the carboxyl-terminal reductase domain of nitric oxide synthase to its heme-containing amino-terminal domain. Available evidence suggests that calmodulin binds to a site between the two domains of nNOS, but it is not known how calmodulin then executes its switch function. To study the calmodulin-nNOS interaction, we created a series of chimeras between calmodulin and cardiac troponin C (cTnC, a homologue of calmodulin that does not activate nNOS). Although a few chimeras showed good ability to activate nNOS, most failed to activate. A subset of the inactive chimeras retained the ability to bind to nNOS and therefore functioned as potent competitive inhibitors of nNOS activation by calmodulin (CaM). The observed inhibition was additive with the arginine antagonists NG-monomethyl-L-arginine and 7-nitroindazole, indicating a distinct and independent mechanism of nNOS inhibition. To localize the calmodulin residues that account for impaired activation in the inhibitory CaM-cTnC chimeras, we conducted a detailed mutagenesis study, replacing CaM subdomains and individual amino acid residues with the corresponding residues from cTnC. This revealed that mutations in CaM helices 2 and 6 (its latch domain) have a disproportionate negative effect on nNOS activation. Thus, our evidence suggests that the CaM latch domain plays a critical role in its molecular switch function.
D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000502:physiology; D004789:Enzyme Activation; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013552:Swine
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase