PDBsum entry 1d0c

Oxidoreductase

PDB id: 1d0c

Contents

Protein chains

416 a.a. *

Ligands

ACT ×4

HEM ×2

INE ×4

CAD ×2

GOL ×2

Metals

_ZN

Waters ×655

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structure of nitric oxide synthase bound to nitro indazole reveals a novel inactivation mechanism.

Authors

C.S.Raman, H.Li, P.Martásek, G.Southan, B.S.Masters, T.L.Poulos.

Ref.

Biochemistry, 2001, 40, 13448-13455. [DOI no: 10.1021/bi010957u]

PubMed id

11695891

Abstract

Nitric oxide is generated under normal and pathophysiological conditions by three distinct isoforms of nitric oxide synthase (NOS). A small-molecule inhibitor of NOS (3-Br-7-nitroindazole, 7-NIBr) is profoundly neuroprotective in mouse models of stroke and Parkinson's disease. We report the crystal structure of the catalytic heme domain of endothelial NOS complexed with 7-NIBr at 1.65 A resolution. Critical to the binding of 7-NIBr at the substrate site is the adoption by eNOS of an altered conformation, in which a key glutamate residue swings out toward one of the heme propionate groups. Perturbation of the heme propionate ensues and eliminates the cofactor tetrahydrobiopterin-heme interaction. We also present three crystal structures that reveal how alterations at the substrate site facilitate 7-NIBr and structurally dissimilar ligands to occupy the cofactor site.

Secondary reference #1

Title

Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center.

Authors

C.S.Raman, H.Li, P.Martásek, V.Král, B.S.Masters, T.L.Poulos.

Ref.

Cell, 1998, 95, 939-950. [DOI no: 10.1016/S0092-8674(00)81718-3]

PubMed id

9875848

Figure 5.
Figure 5. Cooperativity and Molecular Mimicry in eNOS(A) Cross talk between H[4]B and L-Arg mediated by the heme propionate (Se-edge data). The guanidinium and amino groups of L-Arg are held in place by H-bonding with the conserved Glu-363. The amino group also H-bonds with a heme propionate. H[4]B H-bonds directly with the heme propionate, while the pteridine ring is sandwiched between Phe-462 in one monomer and Trp-449 in another, respectively.(B) L-Arg is a structural mimic of H[4]B at the pterin-binding site when SEITU is bound at the active site (-H[4]B, +SEITU data). L-Arg binds to the pterin site and exquisitely mimics the H[4]B interaction with eNOS ([A] and Figure 4). The specific interaction of the potent inhibitor, SEITU, at the active site is mediated by a pair of bifurcated H-bonds to Glu-363. Two water molecules bridge between the inhibitor and heme propionate. The ethyl group of the inhibitor forms nonbonded contacts with Val-338 and Phe-355. The ureido sulfur is positioned 3.5 Å and 4.0 Å above heme pyrrole B-ring nitrogen and the heme iron, respectively.

Figure 7.
Figure 7. Proposed Mechanism for Pterin in NO BiosynthesisThe uniqueness of the H[4]B–eNOS interaction (Figure 4) and the ability to bind L-Arg at the pterin site present a strong case for the involvement of a pterin radical in NOS catalysis and rule out the possibility of H[4]B ↔ qH[2]B cycling during NO biosynthesis. R represents the dihydroxypropyl side chain at the C6 position on the pterin ring.

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #2

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

Secondary reference #3

Title

The structure of nitric oxide synthase oxygenase domain and inhibitor complexes.

Authors

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

Ref.

Science, 1997, 278, 425-431. [DOI no: 10.1126/science.278.5337.425]

PubMed id

9334294

Figure 3.
Fig. 3. Mobility, surface properties, and shape. (A) C trace of NOS[ox] 114 (cubic crystal form) colored by the crystallographic^ temperature factor (low to high B factors colored blue to red) and displayed with heme and mutation sites that affect function. Mutation sites (side chains displayed and labeled by residue number) affecting dimerization, L-Arg binding, or H[4]B binding (defined^ in Fig. 2) cluster to highly mobile (red) projecting regions. The view is rotated by about 45° from Fig. 1 about a vertical axis. (B) Solvent-accessible molecular surface of flattened^ (left) and concave (center) face. The orientation is the same^ as in (A). The exposed heme edge (gold), residues contributing to the distal pocket (cyan), and exposed conserved hydrophobic^ residues (green) (defined in Fig. 2) map to the same flattened^ face of the molecule and cluster in the regions of high mobility and mutational sensitivity shown in (A), making this surface the^ prime candidate for a symmetric dimer interface. (C) Solvent-accessible^ molecular surface of the narrow curved face. This face has few conserved exposed hydrophobic residues. The view is rotated 90° from (A) and (B) around a vertical axis.

Figure 5.
Fig. 5. Comparison of the proximal heme-binding regions of iNOS[ox] and cytochrome P450s. Structural elements contributing to the proximal heme-binding regions of iNOS[ox] 114 and P450[cam] (cyan C traces) are substantially different. Only the proximal Cys ligands (magenta bonds with yellow sulfur atoms, bound to gold^ hemes) and immediately COOH-terminal three residues (magenta C traces) have similar conformations. In iNOS[ox], Cys194 lies at the COOH-terminal end of a helix and precedes an extended^ strand, whereas in P450[cam], Cys357 lies at the NH[2]-terminal end of a helix and follows an extended^ strand. Also, these two cysteine thiolates bind opposite faces of iron protoporphyrin IX. C positions for iNOS[ox] 114 residues 194 to 197 were superimposed with P450[cam] residues 357^ to 360 and then separated for clarity.

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