title: Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase.
abstract: Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.
authors.affiliation: Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA. edg@scripps.edu
authors.forename: Elsa D
authors.initials: ED
authors.lastname: Garcin
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Bruns
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Lloyd
authors.forename: David J
authors.initials: DJ
authors.lastname: Hosfield
authors.forename: Mauro
authors.initials: M
authors.lastname: Tiso
authors.forename: Ratan
authors.initials: R
authors.lastname: Gachhui
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Getzoff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004579:Electron Transport; D005415:Flavins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
chemical_list: D005415:Flavins; D007527:Isoenzymes; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat