title: Inducible nitric oxide synthase: role of the N-terminal beta-hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction.
abstract: The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H(4)B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a beta-hairpin hook, a zinc ligation center and part of the H(4)B-binding site for its role in dimerization, catalysis, and H(4)B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal beta-hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing interactions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H(4)B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H(4)B. Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer.
authors.affiliation: Department of Immunology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: D K
authors.initials: DK
authors.lastname: Ghosh
authors.forename: B R
authors.initials: BR
authors.lastname: Crane
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.forename: D
authors.initials: D
authors.lastname: Wolan
authors.forename: R
authors.initials: R
authors.lastname: Gachhui
authors.forename: C
authors.initials: C
authors.lastname: Crooks
authors.forename: A
authors.initials: A
authors.lastname: Presta
authors.forename: J A
authors.initials: JA
authors.lastname: Tainer
authors.forename: E D
authors.initials: ED
authors.lastname: Getzoff
authors.forename: D J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D019281:Dimerization; D004330:Drosophila; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011622:Pterins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
chemical_list: D011622:Pterins; D011994:Recombinant Proteins; D001708:Biopterins; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; C003402:sapropterin