Nitric Oxide-Induced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in Shewanella oneidensis.
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.
Department of Chemistry, ‡Department of Molecular and Cell Biology, §QB3 Institute, University of California , Berkeley, California 94720, United States.
Minxi
M
Rao
Department of Chemistry, ‡Department of Molecular and Cell Biology, §QB3 Institute, University of California , Berkeley, California 94720, United States.
Mark A
MA
Herzik
Department of Chemistry, ‡Department of Molecular and Cell Biology, §QB3 Institute, University of California , Berkeley, California 94720, United States.
Anthony T
AT
Iavarone
Department of Chemistry, ‡Department of Molecular and Cell Biology, §QB3 Institute, University of California , Berkeley, California 94720, United States.
Michael A
0000-0001-8715-4253
MA
Marletta
D020134:Catalytic Domain / Q000187:drug effects*; D006418:Heme / Q000378:metabolism*; D000071677:Histidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009569:Nitric Oxide / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D020592:Shewanella / Q000201:enzymology*; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
D009569:Nitric Oxide; D006418:Heme; D000071677:Histidine Kinase