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Heme Nitric Oxide/ Oxygen Binding Domains
People working on this project: Mark Herzik
Charles Hespen
Christine Phillips-Piro,Ph.D.
Lars Plate

Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing hemoprotein that has been described in eukaryotes from Drosophila to humans.   Genomic analysis has recently placed sGC within a larger family of proteins with Heme Nitric oxide/Oxygen binding (H-NOX) domains including prokaryotic proteins with significant homology (15-40% identity) to the heme domain of sGC. Predicted H-NOX domains were found in facultative aerobes, obligate anaerobes, and thermophiles. Genomic analysis reveals that the H-NOX domains may be linked to histidine kinases or diguanylate cyclases (obligate anaerobes) or methyl-accepting chemotaxis proteins (obligate anaerobes). Uncovering the biological function of these H-NOX domains is currently an area of intense investigation in our lab.

hnoxstruct

(A) Structural features of Tt H-NOX. (B) Heme binding pocket.

H-NOX proteins also exhibit remarkable diatomic ligand selectivity despite a similar protein fold. For example, the H-NOX domain from Vibrio cholera (a facultative aerobe) binds NO in a high spin 5-coordinate complex and excludes oxygen, while the H-NOX domain from Thermoanaerobacter tengcongensis (Tt, obligate anaerobe) has been found to bind oxygen in a low-spin 6-coordinate complex, making it the first member of the family to bind O2. Current research is focused on understanding the nature of this ligand selectivity from a molecular level and how this selectivity translates into protein function as sensors in biology.

 

Our lab is currently focused on investigating three main areas pertaining to H-NOXs:
1. How do H-NOX domains control ligand affinity and selectivity?
2. How do H-NOX domains regulate their associated signaling proteins?
3. What role do H-NOX domains play in vivo?

A variety of biochemical, structural, and biological methods are being used to investigate these questions.  We use spectroscopic techniques, such as UV-Vis spectroscopy, stopped flow spectroscopy, laser flash photolysis, and resonance Raman spectroscopy, to probe how mutations in the heme pocket affect ligand selectivity and affinity. Both x-ray crystal and NMR solution structural studies are used to determine the effects of heme conformation and ligand binding on the conformation of H-NOX domains. Phosphorylation assays are used to probe the effect of H-NOX redox and ligation state on kinase autophosphorylation activity.

Finally, we are interested in the pathways that are controlled in vivo by H-NOX domains. In facultative aerobes, we postulate that NO produced through denitrification may serve as the signal for low O2 tension.  In obligate anaerobes, we hypothesize that the fusion of the H-NOX to a methyl-accepting chemotaxis protein (MCP) may result in an O2 sensing protein that produces a chemotaxic response away from O2 or as an NO sensor.  We are investigating these hypotheses through biochemical analysis of potential downstream signaling proteins and through the generation of H-NOX knockouts in vivo.

References:

(1)  Weinert EE, Phillips-Piro CM, Tran R, Mathies RA, Marletta MA. Controlling Conformational Flexibility of an O2-binding N-NOX Domain. Biochemistry. 2011 50(32):6832-40..

(2)  Tran R, Weinert EE, Boon EM, Mathies RA, Marletta MA.  Determinants of the Heme-CO Vibrational Modes in the H-NOX Family. Biochemistry. 2011 50(30): 6519-30.

(3)  Olea C, Kuriyan J, Marletta MA.  Modulating heme redox potential through protein-induced porphyrin distortion. J Am Chem Soc. 2010, 132: 12794-5.

(4)  Carlson HK, Vance RE, Marletta MA.  H-NOX regulation of c-di-GMP Metabolism and Biofilm Formation in Legionella pneumophila. Mol Microbiol 2010, 77(4):930-42.

(5)  Wang Y, Dufour YS, Carlson HK, Donohue TJ, Marletta MA, Ruby EG. H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri. Proc Natl Acad Sci USA. 2010, 107: 8375-80.

(6)  Winter MB, McLaurin EJ, Reece SY, Olea C, Nocera DG, Marletta MA. Ru-Porphyrin Protein Scaffolds for Sensing O2. J Am Chem Soc. 2010, 132, 5582-83.

(7) Olea C Jr, Herzik MA Jr, Kuriyan J, Marletta MA. Structural insights into the molecular mechanism of H-NOX activation. Protein Sci. 2010, 19, 881-7.

(8) Weinert EE, Plate L, Whited CA, Olea C Jr, Marletta MA. Determinants of Ligand Affinity and Heme Reactivity in H-NOX Domains. Angew Chem Int Ed Engl. 2010, 49, 720-23.

(9) Erbil WK, Price MS, Wemmer DE, Marletta MA.  A structural basis for H-NOX signaling in Shewanella oneidensis by trapping a histidine kinase inhibitory conformation. Proc Natl Acad Sci USA. 2009, 106, 19753-60.

(10) Carlson HK, Plate L, Price MS, Allen JJ, Shokat KM, Marletta MA.  Use of a semisynthetic epitope to probe histidine kinase activity and regulation. Anal Biochem. 2010, 397, 139-43.

(11) Tran, R; Boon, EM; Marletta, MA; Mathies, RA. Resonance Raman spectra of an O2-binding H-NOX domain reveal heme relaxation upon mutation. Biochemistry 2009, 48, 8568-77.

(12) Olea, C; Boon, EM; Pellicena, P; Kuriyan, J; Marletta, MA. Probing the Function of Heme Distortion in the H-NOX Family. ACS Chem. Biol. 2008, 3, 703-710.

(13) Price, MS; Chao, LY; Marletta, MA. Shewanella oneidensis MR-1 H-NOX Regulation of a Histidine Kinase by Nitric Oxide. Biochemistry 2007, 46, 13677-83.

(14) Boon, EM; Davis, JH; Tran, R; Karow, DS; Huang, SH; Pan, D; Miazgowicz, MM; Mathies, RA; Marletta, MA. Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain. J Biol Chem. 2006, 281, 21892-902.

(15) Boon, EM.; Marletta, MA. Sensitive and Selective Detection of Nitric Oxide Using an H-NOX Domain. J. Am. Chem. Soc. 2006, 128, 10022-3.

(16) Boon, EM; Marletta, MA. Ligand discrimination in soluble guanylate cyclase and the H-NOX family of heme sensor proteins. Curr Opin Chem Biol. 2005, 9, 441-6.

(17) Boon, EM; Huang, SH; Marletta, MA. A molecular basis for NO selectivity in soluble guanylate cyclase. Nat Chem Biol. 2005, 1, 53-9

(18) Boon, EM; Marletta, MA. Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors. J Inorg Biochem. 2005, 99, 892-902.

(19) Karow, DS; Pan, D; Tran, R; Pellicena, P; Presley, A; Mathies, RA; Marletta, MA. Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis. Biochemistry. 2004, 43, 10203-11.

(20) Pellicena, P; Karow, DS; Boon, EM; Marletta, MA; Kuriyan, J. Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases. Proc. Natl. Acad. Sci. USA. 2004, 101, 12854-9.

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