PDBsum entry 1m8h

Oxidoreductase

PDB id: 1m8h

Contents

Protein chains

413 a.a. *

Ligands

SO4

HEM ×2

H4B ×2

6NI ×2

Waters ×89

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Conformational changes in nitric oxide synthases induced by chlorzoxazone and nitroindazoles: crystallographic and computational analyses of inhibitor potency.

Authors

R.J.Rosenfeld, E.D.Garcin, K.Panda, G.Andersson, A.Aberg, A.V.Wallace, G.M.Morris, A.J.Olson, D.J.Stuehr, J.A.Tainer, E.D.Getzoff.

Ref.

Biochemistry, 2002, 41, 13915-13925. [DOI no: 10.1021/bi026313j]

PubMed id

12437348

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.

Secondary reference #1

Title

Structure of nitric oxide synthase oxygenase dimer with pterin and substrate.

Authors

B.R.Crane, A.S.Arvai, D.K.Ghosh, C.Wu, E.D.Getzoff, D.J.Stuehr, J.A.Tainer.

Ref.

Science, 1998, 279, 2121-2126. [DOI no: 10.1126/science.279.5359.2121]

PubMed id

9516116

Figure 1.
Fig. 1. NOS[ox] - fold, dimer assembly, and likely interaction surface for NOS[red] and caveolin. (A) The symmetric iNOS[ox] dimer viewed along the crystallographic twofold axis, showing left (and^ right) subunits with orange (yellow) winged sheets and flanking blue (cyan) helices. Ball-and-stick models (white bonds with red^ oxygen, blue nitrogen, yellow sulfur, and purple iron atoms) highlight active-center hemes (left-most and right-most), interchain disulfide^ bonds (center, foreground), pterin cofactors (white, left-center and right-center), and substrate L-Arg (green left and magenta^ right). The NH[2]-terminal ends contribute hairpins (center top and bottom) to the dimer interface, and the COOH-termini (lower left and upper right) lie 85 Å apart. Gray loops (residues 101^ to 107) are disordered. (B) iNOS[ox] dimer shown rotated^ 90° about a horizontal axis from (A). Each heme is cupped between the inward-facing palm (webbed sheet) and thumb (magenta loop in front of left heme and green loop behind right heme) of the^ "catcher's mitt" subunit fold. (C) Solvent-accessible surface^ (29) of the iNOS[ox] dimer (one subunit red, one subunit blue) oriented as in (B) and color-coded by residue conservation (paler to more saturated represents less conserved to more conserved) in NOS[ox] sequences of known species and isozymes. The heme (white^ tubes) is also solvent-exposed on the side (left subunit) opposite^ the active-center channel (right subunit) and surrounded by a^ highly conserved hydrophobic surface for NOS[red] and caveolin binding. (Stereo variations of Figs.

Figure 5.
Fig. 5. Proposed L-Arg-assisted NOS oxygen activation. First, substrate L-Arg (only guanidinium shown) donates a proton to peroxo-iron, facilitating O-O bond cleavage and conversion to a proposed oxo-iron(IV) -cation radical species, which then rapidly hydroxylates the^ neutral guanidinium to NOH-L-Arg, possibly through a radical-based^ mechanism (3).

The above figures are reproduced from the cited reference with permission from the AAAs