Interaction of CheY2 and CheY2-P with the cognate CheA kinase in the chemosensory-signalling chain of Sinorhizobium meliloti.
An unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti. Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheA-CheY2 and CheA-CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal beta4-alpha4-beta5-alpha5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting beta5 and alpha5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.
Lehrstuhl für Genetik, Universität Regensburg, Regensburg, Germany.
Hubert
H
Riepl
Till
T
Maurer
Hans Robert
HR
Kalbitzer
Veronika M
VM
Meier
Martin
M
Haslbeck
Rüdiger
R
Schmitt
Birgit
B
Scharf
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011494:Protein Kinases