A bridging interaction allows calmodulin to activate NO synthase through a bi-modal mechanism.
Calmodulin (CaM) activates the nitric-oxide synthases (NOS) by a mechanism that is not completely understood. A recent crystal structure showed that bound CaM engages in a bridging interaction with the NOS FMN subdomain. We investigated its importance in neuronal NOS (nNOS) by mutating the two residues that primarily create the bridging interaction (Arg(752) in the FMN subdomain and Glu(47) in CaM). Mutations designed to completely destroy the bridging interaction prevented bound CaM from increasing electron flux through the FMN subdomain and diminished the FMN-to-heme electron transfer by 90%, whereas mutations that partly preserve the interaction had intermediate effects. The bridging interaction appeared to control FMN subdomain interactions with both its electron donor (NADPH-FAD subdomain) and electron acceptor (heme domain) partner subdomains in nNOS. We conclude that the Arg(752)-Glu(47) bridging interaction is the main feature that enables CaM to activate nNOS. The mechanism is bi-modal and links a single structural aspect of CaM binding to specific changes in nNOS protein conformational and electron transfer properties that are essential for catalysis.
Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA.
Jesús
J
Tejero
Mohammad Mahfuzul
MM
Haque
Deborah
D
Durra
Dennis J
DJ
Stuehr
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats
D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D009249:NADP; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I