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

Nature Structural & Molecular Biology volume 11, pages 54–59 (2004)Cite this article

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-Nω-nitroarginine-containing dipeptide inhibitors.

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Figure 1: Ribbon diagram of the eNOS heme domain, the active site and the dipeptide inhibitors used in this study.
Figure 2: Stereo diagrams of the Fo − Fc omit electron density maps contoured at 3 σ of the three dipeptide amide or peptidomimetic inhibitors bound, respectively, to nNOS and eNOS.
Figure 3: Stereo diagrams of dipeptide amide I binding.
Figure 4: Stereo diagrams of the Fo − Fc omit electron density maps contoured at 3 σ of inhibitor I binding.

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Acknowledgements

We thank the beamline staff at SSRL and ALS for their assistance during synchrotron data collection. This research was supported by US National Institutes of Health grants GM57353 (T.L.P) and GM49725 (R.B.S.). J.A.G. acknowledges NATO and the Spanish Ministry of Science and Technology for support.

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Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry and the Program in Macromolecular Structure, University of California, Irvine, 92697-3900, California, USA

    Mack L Flinspach, Huiying Li, Joumana Jamal, Weiping Yang & Thomas L Poulos

  2. Department of Chemistry, Department of Biochemistry, Molecular Biology and Cell Biology, and the Drug Discovery Program, Northwestern University, Evanston, 60208-3113, Illinois, USA

    Hui Huang, Jung-Mi Hah, José Antonio Gómez-Vidal, Elizabeth A Litzinger & Richard B Silverman

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Correspondence to Thomas L Poulos.

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Flinspach, M., Li, H., Jamal, J. et al. Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase. Nat Struct Mol Biol 11, 54–59 (2004). https://doi.org/10.1038/nsmb704

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