Nitric Oxide-Induced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in Shewanella oneidensis

Biochemistry. 2017 Mar 7;56(9):1274-1284. doi: 10.1021/acs.biochem.6b01133. Epub 2017 Feb 21.


Nitric oxide (NO) is implicated in biofilm regulation in several bacterial families via heme-nitric oxide/oxygen binding (H-NOX) protein signaling. Shewanella oneidensis H-NOX (So H-NOX) is associated with a histidine kinase (So HnoK) encoded on the same operon, and together they form a multicomponent signaling network whereby the NO-bound state of So H-NOX inhibits So HnoK autophosphorylation activity, affecting the phosphorylation state of three response regulators. Although the conformational changes of So H-NOX upon NO binding have been structurally characterized, the mechanism of HnoK inhibition by NO-bound So H-NOX remains unclear. In the present study, the molecular details of So H-NOX and So HnoK interaction and regulation are characterized. The N-terminal domain in So HnoK was determined to be the site of H-NOX interaction, and the binding interface on So H-NOX was identified using a combination of hydrogen-deuterium exchange mass spectrometry and surface-scanning mutagenesis. Binding kinetics measurements and analytical gel filtration revealed that NO-bound So H-NOX has a tighter affinity for So HnoK compared that of H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with binding-deficient H-NOX mutants further indicate that while formation of the H-NOX-HnoK complex is required for HnoK to be catalytically active, H-NOX conformational changes upon NO-binding are necessary for HnoK inhibition.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain / drug effects*
  • Heme / metabolism*
  • Histidine Kinase / chemistry*
  • Histidine Kinase / genetics
  • Histidine Kinase / metabolism*
  • Kinetics
  • Models, Molecular
  • Mutagenesis
  • Nitric Oxide / pharmacology*
  • Protein Binding / drug effects
  • Shewanella / enzymology*
  • Signal Transduction / drug effects
  • Substrate Specificity


  • Nitric Oxide
  • Heme
  • Histidine Kinase