title: Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS.
abstract: Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.
authors.affiliation: From the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-2517 and.
authors.forename: Debashree
authors.initials: D
authors.lastname: Basudhar
authors.affiliation: From the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-2517 and.
authors.forename: Yarrow
authors.initials: Y
authors.lastname: Madrona
authors.affiliation: the Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health Science University, Portland, Oregon 97239-3098.
authors.forename: Erik T
authors.initials: ET
authors.lastname: Yukl
authors.affiliation: From the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-2517 and.
authors.forename: Santhosh
authors.initials: S
authors.lastname: Sivaramakrishnan
authors.affiliation: From the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-2517 and.
authors.forename: Clinton R
authors.initials: CR
authors.lastname: Nishida
authors.affiliation: the Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health Science University, Portland, Oregon 97239-3098.
authors.forename: Pierre
authors.initials: P
authors.lastname: Moënne-Loccoz
authors.affiliation: From the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-2517 and ortiz@cgl.ucsf.edu.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D009569:Nitric Oxide; D002248:Carbon Monoxide; C506260:DevS protein, Mycobacterium tuberculosis; D006656:Protamine Kinase
keywords: Mycobacterium tuberculosis; biosensor; carbon monoxide; heme; hydrogen bonding network; molecular dynamics; nitric oxide; oxygen binding; resonance Raman