title: System-level mapping of Escherichia coli response regulator dimerization with FRET hybrids.
abstract: Two-component signal transduction, featuring highly conserved histidine kinases (HKs) and response regulators (RRs), is one of the most prevalent signalling schemes in prokaryotes. RRs function as phosphorylation-activated switches to mediate diverse output responses, mostly via transcription regulation. As bacterial genomes typically encode multiple two-component proteins for distinct signalling pathways, the sequence and structural similarities of RR receiver domains create significant challenges to maintain interaction specificity. It is especially demanding for members of the OmpR/PhoB subfamily, the largest RR subfamily, which share a conserved dimerization interface for phosphorylation-mediated transcription regulation. We developed a strategy to investigate RR interaction by analysing Förster resonance energy transfer (FRET) between cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-fused RRs in vitro. Using the Escherichia coli RR PhoB as a model system, we were able to observe phosphorylation-dependent FRET between fluorescent protein (FP)-PhoB proteins and validated the FRET method by determining dimerization affinity and dimerization-coupled phosphorylation kinetics that recapitulated values determined by alternative methods. Further application of the FRET method to all E. coli OmpR/PhoB subfamily RRs revealed that phosphorylation-activated RR interaction is indeed a common theme for OmpR/PhoB subfamily RRs and these RRs display significant interaction specificity. Weak hetero-pair interactions were also identified between several different RRs, suggesting potential cross-regulation between distinct pathways.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Howard HughesMedical Institute, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Gao
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tao
authors.forename: Ann M
authors.initials: AM
authors.lastname: Stock
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C054562:yellow fluorescent protein, Bacteria; C061278:PhoB protein, Bacteria; D049452:Green Fluorescent Proteins