title: Conformational changes in nitric oxide synthases induced by chlorzoxazone and nitroindazoles: crystallographic and computational analyses of inhibitor potency.
abstract: Nitric oxide is a key signaling molecule in many biological processes, making regulation of nitric oxide levels highly desirable for human medicine and for advancing our understanding of basic physiology. Designing inhibitors to specifically target one of the three nitric oxide synthase (NOS) isozymes that form nitric oxide from the L-Arg substrate poses a significant challenge due to the overwhelmingly conserved active sites. We report here 10 new X-ray crystallographic structures of inducible and endothelial NOS oxygenase domains cocrystallized with chlorzoxazone and four nitroindazoles: 5-nitroindazole, 6-nitroindazole, 7-nitroindazole, and 3-bromo-7-nitroindazole. Each of these bicyclic aromatic inhibitors has only one hydrogen bond donor and therefore cannot form the bidentate hydrogen bonds that the L-Arg substrate makes with Glu371. Instead, all of these inhibitors induce a conformational change in Glu371, creating an active site with altered molecular recognition properties. The cost of this conformational change is approximately 1-2 kcal, based on our measured constants for inhibitor binding to the wild-type and E371A mutant proteins. These inhibitors derive affinity by pi-stacking above the heme and replacing both intramolecular (Glu371-Met368) and intermolecular (substrate-Trp366) hydrogen bonds to the beta-sheet architecture underlying the active site. When bound to NOS, high-affinity inhibitors in this class are planar, whereas weaker inhibitors are nonplanar. Isozyme differences were observed in the pterin cofactor site, the heme propionate, and inhibitor positions. Computational docking predictions match the crystallographic results, including the Glu371 conformational change and inhibitor-binding orientations, and support a combined crystallographic and computational approach to isozyme-specific NOS inhibitor analysis and design.
authors.affiliation: Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB-4, La Jolla, California 92037, USA.
authors.forename: Robin J
authors.initials: RJ
authors.lastname: Rosenfeld
authors.forename: Elsa D
authors.initials: ED
authors.lastname: Garcin
authors.forename: Koustubh
authors.initials: K
authors.lastname: Panda
authors.forename: Gunilla
authors.initials: G
authors.lastname: Andersson
authors.forename: Anders
authors.initials: A
authors.lastname: Aberg
authors.forename: Alan V
authors.initials: AV
authors.lastname: Wallace
authors.forename: Garrett M
authors.initials: GM
authors.lastname: Morris
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Olson
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; D001665:Binding Sites; D002753:Chlorzoxazone / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D006801:Humans; D007191:Indazoles / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000187:drug effects; D008958:Models, Molecular; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000187:drug effects; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009574:Nitro Compounds / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects
chemical_list: D007191:Indazoles; D007527:Isoenzymes; D009125:Muscle Relaxants, Central; D009574:Nitro Compounds; D011994:Recombinant Proteins; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D002753:Chlorzoxazone