The crystal structure of the heme domain of endothelial nitric oxide synthase (eNOS) has been determined to 1.9 ?. NOS is the enzyme that converts L-arginine to nitric axide (NO) and L-citrulline. NO is a ptent biological signaling molecule in the immune, cardiovascular, and nervous systems. Both NO over and under production are associated with a number of pathological conditions so the design of isoform specific inhibitors is an important health related goal. NOS must be dimeric to function and since the heme domain is the critical target for drug design, we have focused on the structure of heme domain dimers. The structure to 1.9 ? contains a potent inhibitor, ehtylisothiourea, bound at the active site and at 2.3 ?, the substrate-complex structure has been determined. The use of synchrotron radiation sources was absolutely essential for this work. Crystals diffract inhouse to about 3.0-3.5 ? but beyond 1.9 ? at synchrotron sources. In addition, extensive use of anomalous dispersion information was essential for obtaining an interpretable electron density map. An unexpected and novel feature in the structure is the identification of metal coordinated by pairs of symmetry related Cys residues at the dimer interface along the dyad axis of symmetry. Since several data sets were obtained at various wavelengths at CHESS and at other sources, it was possible to use anomalous difference Fouriers to identify the metal as zinc. The metal is crucial for forming the binding site for the essential cofactor, tetrhydrobiopterin, which in turn, is important for forming the substrate binding site. The identification of the new metal-S4 cluster has provided significant new insights into NOS structure and function which would not have been possible without synchrotron x-ray sources.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001646-19
Application #
6491146
Study Section
Project Start
2001-08-15
Project End
2002-08-14
Budget Start
Budget End
Support Year
19
Fiscal Year
2001
Total Cost
$142,703
Indirect Cost
Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Kozlov, Guennadi; Wong, Kathy; Gehring, Kalle (2018) Crystal structure of the Legionella effector Lem22. Proteins 86:263-267
Ménade, Marie; Kozlov, Guennadi; Trempe, Jean-François et al. (2018) Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations. J Biol Chem 293:12832-12842
Xu, Jie; Kozlov, Guennadi; McPherson, Peter S et al. (2018) A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy. J Biol Chem 293:4566-4574
Dean, Dexter N; Rana, Pratip; Campbell, Ryan P et al. (2018) Propagation of an A? Dodecamer Strain Involves a Three-Step Mechanism and a Key Intermediate. Biophys J 114:539-549
Chen, Yu Seby; Kozlov, Guennadi; Fakih, Rayan et al. (2018) The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity. J Biol Chem 293:19998-20007
Xu, Caishuang; Kozlov, Guennadi; Wong, Kathy et al. (2016) Crystal Structure of the Salmonella Typhimurium Effector GtgE. PLoS One 11:e0166643
Cogliati, Massimo; Zani, Alberto; Rickerts, Volker et al. (2016) Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population. Fungal Genet Biol 87:22-9
Oot, Rebecca A; Kane, Patricia M; Berry, Edward A et al. (2016) Crystal structure of yeast V1-ATPase in the autoinhibited state. EMBO J 35:1694-706
Lucido, Michael J; Orlando, Benjamin J; Vecchio, Alex J et al. (2016) Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry 55:1226-38
Bauman, Joseph D; Harrison, Jerry Joe E K; Arnold, Eddy (2016) Rapid experimental SAD phasing and hot-spot identification with halogenated fragments. IUCrJ 3:51-60

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