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Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation

Nature Structural Biology volume 6, pages 233–242 (1999)Cite this article

Abstract

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 Å resolution) and iNOS (2.25 Å resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

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Figure 1: Electron density maps in the immediate vicinity of the zinc (a,b ) or BH4 (c,d), contoured at 1.2σ (green) and 3.6σ (purple).
Figure 2: Structures of a, eNOSox and b, iNOSox monomers shown as ribbon diagrams, along with heme, BH4 and either the arginine substrate for eNOSox or the inhibitor SEITU for iNOSox, drawn in a ball-and-stick representation.
Figure 3: Secondary structure and sequence alignment of NOS oxygenase domains.
Figure 4: Stereo view of the eNOSox and iNOSox dimerization interface.
Figure 5: Comparison of the eNOS and iNOS catalytic sites.
Figure 6: View of the iNOSox interdomain 'hook'.

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References

  1. Stuehr, D.J. Structure–function aspects in the nitric oxide synthases. Annu. Rev. Pharmacol. Toxicol. 37, 339– 59 (1997).

    Article  CAS  Google Scholar 

  2. Nathan, C. Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest. 100, 2417–2423 (1997).

    Article  CAS  Google Scholar 

  3. Nakane, M. et al. Novel potent and selective inhibitors of inducible nitric oxide synthase. Mol. Pharmacol. 47, 831– 834 (1995).

    CAS  PubMed  Google Scholar 

  4. Crane, B.R. et al. The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science 278, 425– 431 (1997).

    Article  CAS  Google Scholar 

  5. Crane, B.R. et al. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279, 2121– 2126 (1998).

    Article  CAS  Google Scholar 

  6. Geller, D.A. et al. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90, 3491–3495 ( 1993).

    Article  CAS  Google Scholar 

  7. Garvey, E.P. et al. Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas. J Biol. Chem. 269, 26669–26676 (1994).

    CAS  PubMed  Google Scholar 

  8. Janssens, S.P., Simouchi, A., Quertermous, T., Bloch, D.B. & Bloch, K.D. Cloning and expression of a cDNA encoding human endothelium-derived relating factor/nitric oxide synthase. J. Biol. Chem. 267, 22694 ( 1992).

    CAS  PubMed  Google Scholar 

  9. Deisenhofer, J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20, 2361–2370 (1981).

    Article  CAS  Google Scholar 

  10. Tzeng, E., Billiar, T.R., Robbins, P.D., Loftus, M. & Stuehr, D.J. Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer. Proc. Natl. Acad. Sci. U.S.A. 92, 11771–11775 (1995).

    Article  CAS  Google Scholar 

  11. Chen, P.F., Tsai, A.L. & Wu, K.K. Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity. Biochem. Biophys. Res. Commun. 215, 1119 –1129 (1995).

    Article  CAS  Google Scholar 

  12. Chen, P.F., Tsai, A.L. & Wu, K.K. Cysteine 184 of endothelial nitric oxide synthase is involved in heme coordination and catalytic activity. J. Biol. Chem. 269, 25062–25066 (1994).

    CAS  PubMed  Google Scholar 

  13. Ghosh, D.K. et al. Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction. Biochemistry 36 , 10609–10619 (1997).

    Article  CAS  Google Scholar 

  14. Vallee, B.L. & Auld, D.S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647–5659 (1990).

    Article  CAS  Google Scholar 

  15. Karlin, S. & Zhu, Z.Y. Classification of mononuclear zinc metal sites in protein structures. Proc. Natl. Acad. Sci. U.S.A. 94, 14231–14236 ( 1997).

    Article  CAS  Google Scholar 

  16. Cameron, A.D., Olin, B., Ridderstrom, M., Mannervik, B. & Jones, T.A. Crystal structure of human glyoxalase I—evidence for gene duplication and 3D domain swapping. Embo J. 16, 3386–3395 ( 1997).

    Article  CAS  Google Scholar 

  17. Ren, B. et al. A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units. Nature Struct. Biol. 5, 602–611 ( 1998).

    Article  CAS  Google Scholar 

  18. Radhakrishnan, R. et al. Zinc mediated dimer of human interferon-alpha 2b revealed by X-ray crystallography. Structure 4, 1453 –1463 (1996).

    Article  CAS  Google Scholar 

  19. Wang, J., Stuehr, D.J., Ikeda-Saito, M. & Rousseau, D.L. Heme coordination and structure of the catalytic site in nitric oxide synthase. J. Biol. Chem. 268, 22255– 22258 (1993).

    CAS  PubMed  Google Scholar 

  20. Cubberley, R.R. et al. Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem, nitroarginine and tetrahydrobiopterin. Biochem. J. 323, 141 –146 (1997).

    Article  CAS  Google Scholar 

  21. McMillan, K. & Masters, B.S. Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415. Biochemistry 34, 3686–3693 (1995).

    Article  CAS  Google Scholar 

  22. Sari, M.A. et al. Expression in yeast and purification of functional macrophage nitric oxide synthase. Evidence for cysteine-194 as iron proximal ligand. Biochemistry 35, 7204– 7213 (1996).

    Article  CAS  Google Scholar 

  23. Bolton, W. & Perutz, M.F. Three dimensional Fourier synthesis of horse deoxyhaemoglobin at 2.8 Å resolution. Nature 228, 551–552 (1970).

    Article  CAS  Google Scholar 

  24. Chen, P.F., Tsai, A.L., Berka, V. & Wu, K.K. Mutation of Glu-361 in human endothelial nitric-oxide synthase selectively abolishes l-arginine binding without perturbing the behavior of heme and other redox centers. J. Biol. Chem. 272, 6114–6118 (1997).

    Article  CAS  Google Scholar 

  25. Gachhui, R. et al. Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding. Biochemistry 36, 5097– 5103 (1997).

    Article  CAS  Google Scholar 

  26. Stuehr, D.J. et al. N omega-hydroxy-l-arginine is an intermediate in the biosynthesis of nitric oxide from l-arginine. J. Biol. Chem. 266 , 6259–6263 (1991).

    CAS  PubMed  Google Scholar 

  27. Macdonald, J.E. In Nitric oxide synthase inhibitors (ed. Bristol, J.A.) 221 –230 (Academic Press, Inc., San Diego; 1996).

    Google Scholar 

  28. Mayer, B. et al. Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin. Biochemistry 36, 8422–8427 (1997).

    Article  CAS  Google Scholar 

  29. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–325 ( 1997).

    Article  CAS  Google Scholar 

  30. Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760– 763 (1994).

    Google Scholar 

  31. Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–493 (1997).

    Article  Google Scholar 

  32. Abrahams, J.P. & Leslie, A.G.W. Methods used in the structure determination of bovine mitochondrial F~1 ATPase. Acta Crystallogr. D 52, 30–42 (1996).

    Article  CAS  Google Scholar 

  33. Cowtan, K. & Main, P. Miscellaneous algorithms for density modification. Acta Crystallogr. D 54, 487 –493 (1998).

    Article  CAS  Google Scholar 

  34. Navaza, J. & Saludjian, P. AMoRe: an automated molecular replacement program package. Methods Enzymol. 581– 593 (1997).

  35. Bricogne, G. Bayesian statistical viewpoint on structure determination: basic concepts and examples. Meth. Enz., 276, 361– 423 (1997).

    Article  CAS  Google Scholar 

  36. Brünger, A.T., Krukowski, A. & Erickson, J.W. Slow-cooling protocols for crystallographic refinement by simulated annealing. Acta Crystallogr A 46, 585–593 (1990).

    Article  Google Scholar 

  37. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr 26, 283– 291 (1993).

    Article  CAS  Google Scholar 

  38. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

    Article  CAS  Google Scholar 

  39. Alexandrov, N.N. & Fisher, D. Analysis of topological and nontopological structural similarities in the PDB: new examples with old structures. Proteins 25, 354– 365 (1996).

    Article  CAS  Google Scholar 

  40. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946 –950 (1991).

    Article  Google Scholar 

  41. Merritt, E.A. & Bacon, D.J. Raster3D: photorealistic molecular graphics. Meth. Enz. 277, 505– 524 (1997).

    Article  CAS  Google Scholar 

  42. Wallace, A.C., Laskowski, R.A. & Thornton, J.M. LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions. Protein Engng. 8, 127–134 (1995).

    Article  CAS  Google Scholar 

  43. Klegwegt, G.J. & Brunger, A.T. Checking your imagination: application of the free R value. Structure 4, 897–904 (1996).

    Article  Google Scholar 

Download references

Acknowledgements

We thank G. Hammond for technical assistance, C. Eckhart for carrying the atomic absorption experiments, Y.-H. Liu and B. Pramanik for the mass spectrometry analyses, and G. Bricogne, P. Reversi and C. Strickland for helpful discussions.

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Authors and Affiliations

  1. Structural Chemistry Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, 07033, New Jersey, USA

    Thierry O. Fischmann, Alan Hruza, Andrew J. Prongay, Paul Reichert & Patricia C. Weber

  2. Immunology Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, 07033, New Jersey, USA

    Xiao Da Niu, James D. Fossetta, Charles A. Lunn, Edward Dolphin, Daniel J. Lundell & Satwant K. Narula

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  1. Thierry O. Fischmann
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Correspondence to Thierry O. Fischmann.

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Fischmann, T., Hruza, A., Niu, X. et al. Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation. Nat Struct Mol Biol 6, 233–242 (1999). https://doi.org/10.1038/6675

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