PDBsum entry 1p6h

Oxidoreductase

PDB id: 1p6h

Contents

Protein chains

407 a.a. *

Ligands

ACT ×2

HEM ×2

H4B ×2

DP1 ×2

Metals

_ZN

Waters ×487

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural basis for dipeptide amide isoform-Selective inhibition of neuronal nitric oxide synthase.

Authors

M.L.Flinspach, H.Li, J.Jamal, W.Yang, H.Huang, J.M.Hah, J.A.Gómez-Vidal, E.A.Litzinger, R.B.Silverman, T.L.Poulos.

Ref.

Nat Struct Mol Biol, 2004, 11, 54-59. [DOI no: 10.1038/nsmb704]

PubMed id

14718923

Abstract

Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.

Figure 1.
Figure 1. Ribbon diagram of the eNOS heme domain, the active site and the dipeptide inhibitors used in this study. (a) Chemical structures of the three dipeptide amide or peptidomimetic NOS inhibitors used in this study: I, L-N^ -nitroarginine-2,4- L-diaminobutyramide; II (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine; III, L-N^ -nitroarginine-(4R)-amino-L-proline amide. (b) Ribbon diagram of eNOS heme domain. All three isoforms share the similar dimeric fold and have a wide open solvent-accessible channel connecting the heme active site to the molecular surface. (c) L-NNA bound in the active site of eNOS. The extensive hydrogen bonding network (dashed lines) between L-NNA and enzyme may explain its low-nanomolar potency. The active site structure and interactions between L-arginine and the protein are the same in all three mammalian NOS isoforms. The only exception is Asn368, which is aspartate in nNOS and iNOS. Even so, the aspartate and asparagine side chains are oriented in the same way in all three structures.

Figure 4.
Figure 4. Stereo diagrams of the F[o] - F[c] omit electron density maps contoured at 3 of inhibitor I binding. (a,b) Inhibitor I bound to nNOS D597N mutant (a) and eNOS N368D mutant (b). In b, the curled binding mode was observed at 70% occupancy, with the wild-type binding mode present at 30% (data not shown). Occupancies were empirically determined by adjusting occupancies of the two alternate conformations until F[o] - F[c] electron density maps indicated no further changes were required. For nNOS only one conformation was observed.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2004, 11, 54-59) copyright 2004.

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

The novel binding mode of n-Alkyl-N'-Hydroxyguanidine to neuronal nitric oxide synthase provides mechanistic insights into no biosynthesis.

Authors

H.Li, H.Shimizu, M.Flinspach, J.Jamal, W.Yang, M.Xian, T.Cai, E.Z.Wen, Q.Jia, P.G.Wang, T.L.Poulos.

Ref.

Biochemistry, 2002, 41, 13868-13875. [DOI no: 10.1021/bi020417c]

PubMed id

12437343