Distal hydrogen-bonding interactions in ligand sensing and signaling by Mycobacterium tuberculosis DosS

Debashree Basudhar, Yarrow Madrona, Erik T. Yukl, Santhosh Sivaramakrishnan, Clinton R. Nishida, Pierre Moenne-Loccoz, Paul R. Ortiz De Montellano

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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.

Original languageEnglish (US)
Pages (from-to)16100-16111
Number of pages12
JournalJournal of Biological Chemistry
Volume291
Issue number31
DOIs
StatePublished - Jul 29 2016

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ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Basudhar, D., Madrona, Y., Yukl, E. T., Sivaramakrishnan, S., Nishida, C. R., Moenne-Loccoz, P., & Ortiz De Montellano, P. R. (2016). Distal hydrogen-bonding interactions in ligand sensing and signaling by Mycobacterium tuberculosis DosS. Journal of Biological Chemistry, 291(31), 16100-16111. https://doi.org/10.1074/jbc.M116.724815