title: Interaction Analysis of a Two-Component System Using Nanodiscs.
abstract: Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems.
authors.affiliation: Fachbereich Biologie/Chemie, Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, D-49076, Osnabrück, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Hörnschemeyer
authors.affiliation: Fachbereich Biologie/Chemie, Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, D-49076, Osnabrück, Germany.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Liss
authors.affiliation: Munich Center for Integrated Protein Science (CiPSM) at the Department of Microbiology, Ludwig-Maximilians-Universität München, 82152, Martinsried, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Heermann
authors.affiliation: Munich Center for Integrated Protein Science (CiPSM) at the Department of Microbiology, Ludwig-Maximilians-Universität München, 82152, Martinsried, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jung
authors.affiliation: Fachbereich Biologie/Chemie, Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, D-49076, Osnabrück, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Hunke
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D000071677:Histidine Kinase; D008051:Lipid Bilayers / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase