Summary of Research Experience
W. Kaya Erbil
Graduate research, University of California, Berkeley
I. Structural consequences of axial histidine-iron bond cleavage in a selective nitric oxide sensor
Principle investigators Michael A. Marletta1,2,3,4,5 and David E. Wemmer1,3,4,5
Other Contributors Mark P. Price2,3,4,5, Jeffery G. Pelton4,5
Department affiliations 1Department of Chemistry, 2Department of Molecular and Cellular Biology, 3QB3 Institute, 4Division of Physical Biosciences, 5Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720-3220, USA
Nitric oxide (NO) signaling in eukaryotes involves activation of soluble guanylate cyclase (sGC) (1). Prokaryotic homologues of the sGC heme domain, termed H-NOX (heme nitric oxide and/or oxygen binding domain) proteins, have been discovered (2, 3). The H-NOX gene in Shewanella oneidensis (S.o.) is within a predicted operon in tandem with a histidine kinase (4). Like sGC, this H-NOX (SO2144) forms a 5-coordinate NO complex and shows no measurable affinity for O2. The NO-bound H-NOX inhibits the constitutively active S.o. histidine kinase (SO2145), whereas the ligand-free protein has no effect, suggesting that the H-NOX and histidine kinase function together in a NO responsive two-component signaling pathway (4). We have determined the solution NMR structure of the S.o. H-NOX domain in complex with the heme ligand carbon monoxide (CO). This complex has kinase inhibitory activity intermediate between ligand free and NO bound protein and forms a diamagnetic state, which can be probed by NMR. The mutant H103G, which requires imidazole to rescue heme binding, also binds CO to form a diamagnetic state. This mutant has activity similar to that of wild type NO complex. The solution NMR structure of this active H-NOX conformation (kinase inactive) has also been determined. Comparison of these two structures reveals ligand induced conformational changes that may be involved in kinase inhibition.
1. W. Kaya Erbil, Mark P. Price, Jeffery G. Pelton, David E. Wemmer, Michael A. Marletta, “Structural Consequences of Axial histidine-iron bond cleavage in a selective nitric oxide sensor.” Manuscript in preparation.
ï Developed a nuclear magnetic resonance (NMR) protocol for high-resolution heme protein structure determination
ï Prepared and structurally analyzed different ligation states of H-NOX
ï Interpreted in vitro kinetic assays to evaluate H-NOX activity, in collaboration with
II. Biological activity and solution structure of the apo-dinitrogenase and FeMo-co interacting domains of NafY
Principle investigators Luis Rubio2 and David E. Wemmer1,3,4,5
Other Contributors Aaron P. Phillips1,3,4,5, Jose Hernandez2, Jeffery G. Pelton4,5
Department affiliations 1Departmentof Chemistry, 2Department of Plant and Microbial Biology, 3QB3 Institute, 4Division of Physical Biosciences, 5Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720-3220, USA
Nitrogen fixation is the conversion of N2 gas into ammonia (5). Most biological nitrogen fixation is carried out by molybdenum nitrogenases present in a select set of microorganisms (6). The active site of these nitrogenases contains the iron-molybdenum cofactor (FeMo-co), whose synthesis involves the function of more than twenty genes: enzymes, molecular scaffolds, and carriers (7). NafY is an escort protein shown to participate in the final steps of FeMo-co synthesis, by interacting with both FeMo-co and the apo-dinitrogenase (8). NafY, a two-domain protein, has an N-terminal domain of unknown function (7) and a C-terminal FeMo-co-interaction domain (9). In this work, we perform a complete biochemical and structural characterization of both domains of the NafY protein to investigate the molecular structure of the apo-dinitrogenase and the FeMo-co-interacting domains of NafY. We demonstrate that the N-terminal domain of NafY displays an apo-dinitrogenase binding activity by itself. Moreover, by tracking the EPR signature of FeMo-co, we show that the binding of the N-terminal domain of NafY to apo-dinitrogenase prevents the insertion of FeMo-co. We solved the solution structure of the N-terminal domain of NafY and found that this structural motif belongs to the SAM family of protein-protein interaction domains. Furthermore, chemical shift perturbation NMR analyses of the core domain of NafY bound to FeMo-co suggest the region of interaction of the cofactor with this protein domain. Altogether, these results support a role for NafY in the delivery of FeMo-co to the apo-dinitrogenase and reveal new insights to the mechanism of cofactor insertion.
2. Aaron P. Phillips*, W. Kaya Erbil*, Jose Hernandez*, Jeffery G. Pelton, Luis Rubio, David E. Wemmer, “Biological activity and solution structure of the apo-dinitrogenase and FeMo-co interacting domains of NafY.” Manuscript in preparation. (* Equal contribution)
ï Used NMR spectroscopy to study the structure and function of metallocluster chaperones, in collaboration with Phillips and Hernandez
Undergraduate research, Emory University, Atlanta, GA
III. Semagenesis and the parasitic angiosperm Striga asiatica
Principle investigator David G. Lynn1,2
Other Contributors William J. Keyes1, Andew G. Palmer1,2, Jeannette V. Taylor3, Robert P. Apkarian3, Eric R. Weeks4
Department affiliations 1Department of Chemistry and Biology, 2Center for Fundamental and Applied Molecular Evolution, 3Integrated Microscopy and Microanalytical Facility/Department of Chemistry, and 4Department of Physics, Emory University, Atlanta, GA 30322, USA
Over the last several years, intermediates in the reduction of dioxygen have been attributed diverse functional roles ranging from protection against pathogen attack to the regulation of cellular development. Evidence now suggests that parasitic angiosperms, which naturally commit to virulence through the growth of new organs, depend on reduced oxygen intermediates, or reactive oxygen species (ROS), for signal generation. Clearly, the role of ROS in both plant defense and other physiological responses complicates any models that employ these intermediates in host plant recognition. We exploited the transparent young Striga asiatica seedling to (i) localize the site of H2O2 accumulation to the surface cells of the primary root meristem, (ii) demonstrate the accumulation of H2O2 within cytoplasmic and apoplastic compartments, and (iii) document precise regulation of H2O2 accumulation during development of the host attachment organ, the haustorium. These studies revealed a new active process for signal generation, host detection and commitment that is capable of ensuring the correct spatial and temporal positioning for attachment.
3. William J. Keyes*, Andrew G. Palmer*, W. Kaya Erbil, Jenette V. Taylor, Robert P. Apkarian, Eric R. Weeks, David G. Lynn, “Semagenesis and the Parasitic Angiosperm Striga asiatica,” The Plant Journal 2007, 51, 707-716. (* Equal contribution)
4. Andrew G. Palmer, Rong Gao, Justin Maresh, W. Kaya Erbil, David G. Lynn, “Chemical Biology Multi-Host/Pathogen Interactions: Chemical Perception and Metabolic of Multi-Host/Pathogen Interactions: Chemical Perception and Metabolic Complementation,” Annu. Rev. Phytopathol., 2004, 42, 439-464.
5. William J. Keyes, David G. Lynn, W. Kaya Erbil, Jenette V. Taylor, Robert P. Apkarian, “H2O2 in Interspecies Signaling: A New Role in Host Detection,” Microsc. Microanal., 2002, 8 (Suppl. 2), 962CD.
ï Monitored the production of ROS by electron and confocal microscopy
ï Studied developmental process in Striga asiatica using chemical modulators
1. Denninger, J.W. and Marletta, M.A. (1999). Guanylate cyclase and the NO/cGMP signaling pathway. Biochimica et Biophysica Acta 1411:334-350.
2. Iyer, L.M., Anantharaman, V., and Aravind, L. (2003). Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins. BMC Genomics 4:5.
3. Boon, E.M. and Marletta, M.A. (2005). Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors. J. Inorg. Biochem. 99:892-902.
4. Price, M.S., Chao, L.Y., and Marletta, M.A. (2007). Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide. Biochemistry 46:13677-13683.
5. Howard, J.B. and Rees, D.C. (2006). How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation. Proc. Natl. Acad. Sci. U S A 103:17088-17093.
6. Dixon, R. and Kahn, D. (2004). Genetic regulation of biological nitrogen fixation. Nat. Rev. Microbiol. 2:621-631.
7. Rubio, L.M. and Ludden, P.W. (2008). Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu. Rev. Microbiol. 62:93-111.
8. Curatti, L., et al. (2007). In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins. Proc. Natl. Acad. Sci. U S A 104:17626-17631.
9. Dyer, D.H., et al. (2003). The three-dimensional structure of the core domain of Naf Y from Azotobacter vinelandii determined at 1.8-A resolution. J. Biol. Chem. 278:32150-32156.