Structural characterization of the heme-based oxygen sensor, AfGcHK, its interactions with the cognate response regulator, and their combined mechanism of action in a bacterial two-component signaling system.
The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.
Martin
M
Stranava
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.|Department of Chemical Education, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.
Václav
V
Martínek
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.|Laboratory of Structure Biology and Cell Signalling, BioCeV - Institute of Microbiology, Czech Academy of Sciences, V.V.I, Prumyslova 595, Vestec, Czech Republic.
Petr
P
Man
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.
Veronika
V
Fojtikova
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.|Laboratory of Structure Biology and Cell Signalling, BioCeV - Institute of Microbiology, Czech Academy of Sciences, V.V.I, Prumyslova 595, Vestec, Czech Republic.
Daniel
D
Kavan
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.
Ondřej
O
Vaněk
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic.
Toru
T
Shimizu
Department of Biochemistry, Charles University in Prague, Hlavova (Albertov) 2030-8, Prague 2, Czech Republic. marketa.martinkova@natur.cuni.cz.
Marketa
0000-0002-0865-8785
M
Martinkova
D048409:Aeromonas salmonicida / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D000071677:Histidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D009231:Myxococcales / Q000737:chemistry* / Q000201:enzymology; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006418:Heme; D006639:Histidine; D007501:Iron; D000071677:Histidine Kinase; D010100:Oxygen
analytical ultracentrifugation; heme-based oxygen sensor; histidine kinase; homology modeling; hydrogen-deuterium exchange; protein-protein docking; protein-protein interaction; two-component signal transduction system