Nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a diffusible messenger for platelet aggregation, blood pressure regulation, neurotransmission, long-term potentiation, and also as a cytotoxic agent for defense against tumor cells and parasites. Three NOS enzymes, the inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) isoforms, achieve their key biological functions via intriguing regulations of their electron transfer mechanism and an assembly of six cofactors. Each subunit of the NOS dimer has two modules joined by a calmodulin-binding linker: an oxygenase module (NOSox) with heme, tetrahydrobiopterin (H4B), Zn ion, and Arginine binding sites forming the catalytic center for NO production, and a reductase module (NOSred) with NADPH, FAD, and FMN sites supplying electrons to the heme. Our overall goal is to characterize the detailed structural biochemistry underlying the active site interactions, catalysis, isozyme-specificity, assembly, regulation, and both inter-domain and inter-protein interactions of NOS enzymes. Our characterizations of the independently functional dimeric NOSox and NOSred modules, and of calmodulin (CaM) bound to the CaM-binding peptide, provide a powerful framework for interpreting NOS structural biochemistry. Our progress to date prompts four proposed Aims, which are driven by specific hypotheses. We now propose integrated structural, mutational and biophysical experiments to test these hypotheses and to address specific critical and challenging unanswered questions. How do NOSox, NOSred and CaM assemble for function? What is the mechanism for rate-limiting electron transfer from the NOSred FMN to the NOSox heme? How do isozyme-specific features tune and regulate NOS activity? How is NOS activity regulated through interactions with its protein partners? Our interdisciplinary experiments on NOS domains and full-length proteins will characterize active-site interactions, key assemblies, conformational switching mechanisms, and inter-protein interactions. We expect to characterize prototypical sets of structures and mutant enzymes, functional complexes with inhibitors and with protein partners, and to define the structural chemistry underlying the exquisite regulation of NO( synthesis. Deuterium Hydrogen Exchange Mass Spectrometry (DXMS) and advanced Small-Angle X- ray Scattering (SAXS) combined with computationally-aided design of mutants to lock, strengthen or block interactions (including designed disulfide linkages) will test and complement high resolution crystallographic structures. The expected outcome of the proposed research is a detailed molecular understanding of the activity, inhibition, and regulation of NOS isozymes relevant to important aspects of their biology and medical importance for blood pressure regulation, stroke, septic shock, cancer and inflammatory damage.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058883-13
Application #
8013576
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Charette, Marc F
Project Start
1997-08-15
Project End
2013-01-31
Budget Start
2011-02-01
Budget End
2013-01-31
Support Year
13
Fiscal Year
2011
Total Cost
$481,355
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Haque, Mohammad Mahfuzul; Bayachou, Mekki; Fadlalla, Mohammed A et al. (2013) Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase. Biochem J 450:607-17
Haque, Mohammad Mahfuzul; Tejero, Jesús; Bayachou, Mekki et al. (2013) Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis. FEBS J 280:4439-53
Nagpal, Latika; Haque, Mohammad M; Saha, Amit et al. (2013) Mechanism of inducible nitric-oxide synthase dimerization inhibition by novel pyrimidine imidazoles. J Biol Chem 288:19685-97
Haque, Mohammad Mahfuzul; Fadlalla, Mohammed A; Aulak, Kulwant S et al. (2012) Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains. J Biol Chem 287:30105-16
Haque, Mohammad M; Kenney, Claire; Tejero, Jesús et al. (2011) A kinetic model linking protein conformational motions, interflavin electron transfer and electron flux through a dual-flavin enzyme-simulating the reductase activity of the endothelial and neuronal nitric oxide synthase flavoprotein domains. FEBS J 278:4055-69
Rosenfeld, Robin J; Bonaventura, Joseph; Szymczyna, Blair R et al. (2010) Nitric-oxide synthase forms N-NO-pterin and S-NO-cys: implications for activity, allostery, and regulation. J Biol Chem 285:31581-9
Guan, Zhi-Wen; Haque, Mohammad Mahfuzul; Wei, Chin-Chuan et al. (2010) Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer. J Biol Chem 285:3064-75
Haque, Mohammad Mahfuzul; Fadlalla, Mohammed; Wang, Zhi-Qiang et al. (2009) Neutralizing a surface charge on the FMN subdomain increases the activity of neuronal nitric-oxide synthase by enhancing the oxygen reactivity of the enzyme heme-nitric oxide complex. J Biol Chem 284:19237-47
Garcin, Elsa D; Arvai, Andrew S; Rosenfeld, Robin J et al. (2008) Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat Chem Biol 4:700-7
Wei, Chin-Chuan; Wang, Zhi-Qiang; Tejero, Jesus et al. (2008) Catalytic reduction of a tetrahydrobiopterin radical within nitric-oxide synthase. J Biol Chem 283:11734-42

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