A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB

Creager-Allen, Rachel L.; Silversmith, Ruth E.; Bourret, Robert B.

doi:10.1074/jbc.m113.471763

Cited by 40 publications

(54 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Plasmids encoding His 6 -tagged CheY variants were made using QuikChange (Agilent) with plasmid pKC1 (14) as the template. CheA was purified as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

“…The additional GSH does not affect CheY autodephosphorylation kinetics (5). Plasmids encoding His 6 -tagged PhoB 1-127 variants used pET28a-phoB N (14), which encodes a thrombincleavable His 6 -tagged PhoB , as a template. Thrombin cleavage leaves the N-terminal GSH.…”

Section: Methodsmentioning

confidence: 99%

“…PhoB variants were purified as described for CheY. His 6 -tagged PhoR 193-431 was expressed and purified as described previously (14). All protein variants were gel filtered using a Superdex 75 1660 size exclusion column (GE Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…E. coli PhoB N , a receiver domain with a relatively low autodephosphorylation rate constant (14), was used to analyze substitutions that could potentially enhance autodephosphorylation. This expands a previous approach using the B. subtilis Spo0F response regulator (11).…”

Section: Amino Acids At Receiver Domain Positions D ؉ 2 and T ؉ 2 Havmentioning

confidence: 99%

See 3 more Smart Citations

Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics

Page

Immormino

et al. 2016

J Bacteriol

Self Cite

View full text Add to dashboard Cite

Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to environmental stimuli. TCSs mediate responses through phosphotransfer from a conserved histidine on a sensor kinase to a conserved aspartate on the receiver domain of a response regulator. Typically, signal termination occurs through dephosphorylation of the receiver domain, which can catalyze its own dephosphorylation. Despite strong structural conservation between receiver domains, reported autodephosphorylation rate constants (k dephos ) span a millionfold range. Variable receiver domain active-site residues D ؉ 2 and T ؉ 2 (two amino acids C terminal to conserved phosphorylation site and Thr/Ser, respectively) influence k dephos values, but the extent and mechanism of influence are unclear. We used sequence analysis of a large database of naturally occurring receiver domains to design mutant receiver domains for experimental analysis of autodephosphorylation kinetics. When combined with previous analyses, k dephos values were obtained for CheY variants that contained D ؉ 2/T ؉ 2 pairs found in 54% of receiver domain sequences. Tested pairs of amino acids at D ؉ 2/T ؉ 2 generally had similar effects on k dephos in CheY, PhoB N , or Spo0F. Acid or amide residues at D ؉ 2/T ؉ 2 enhanced k dephos . CheY variants altered at D ؉ 2/T ؉ 2 exhibited rate constants for autophosphorylation with phosphoramidates and autodephosphorylation that were inversely correlated, suggesting that D ؉ 2/T ؉ 2 residues interact with aspects of the ground or transition states that differ between the two reactions. k dephos of CheY variants altered at D ؉ 2/T ؉ 2 correlated significantly with k dephos of wild-type receiver domains containing the same D ؉ 2/T ؉ 2 pair. Additionally, particular D ؉ 2/T ؉ 2 pairs were enriched in different response regulator subfamilies, suggesting functional significance. T wo-component systems (TCSs) are a prevalent means of signal transduction used by plants and microorganisms to mediate responses to stimuli (1). TCSs are present in more than 95% of sequenced bacterial genomes (2, 3), and one species can contain tens to more than a hundred TCSs. TCSs regulate a wide range of processes from cell development to virulence. Signal transduction by TCSs occurs through the transfer of phosphoryl groups between histidyl and aspartyl residues of different protein components (4). Canonically, the sensory component (the sensor kinase) is a phosphodonor to the response regulator (the response-mediating component) (4). The conserved domain in the response regulator, the receiver domain, functions as a molecular switch. The phosphorylation status of a conserved Asp on the receiver domain corresponds to turning the output response on and off. Typically, receiver domain phosphorylation initiates the output response, and dephosphorylation terminates the response. Dephosphorylation can occur with the assistance of another protein, such as a phosphatase, or by self-catalysis by the receiver domain, which is term...

show abstract

“…Plasmids encoding His 6 -tagged CheY variants were made using QuikChange (Agilent) with plasmid pKC1 (14) as the template. CheA was purified as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Amino Acids At Receiver Domain Positions D ؉ 2 and T ؉ 2 Havmentioning

confidence: 99%

See 2 more Smart Citations

Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics

Page

Immormino

et al. 2016

J Bacteriol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Dimerization of OmpR after phosphorylation is the driving force for phosphorylation-mediated regulation of OmpR-DNA binding (Barbieri et al 2013). However, a previous report noted that a heterodimer (one phosphorylated and one unphosphorylated monomer) can enhance autophosphorylation of RR PhoB and might participate in gene regulation (Creager-Allen et al 2013). Therefore, gene regulation of the OmpR/PhoB family might not require dimerization with phosphorylated monomers.…”

Section: Phosphorylation-dependent Activationmentioning

confidence: 96%

Prokaryotic 2-component systems and the OmpR/PhoB superfamily

et al. 2015

View full text Add to dashboard Cite

Abstract:In bacteria, 2-component regulatory systems (TCSs) are the critical information-processing pathways that link stimuli to specific adaptive responses. Signals perceived by membrane sensors, which are generally histidine kinases, are transmitted by response regulators (RRs) to allow cells to cope rapidly and effectively with environmental challenges. Over the past few decades, genes encoding components of TCSs and their responsive proteins have been identified, crystal structures have been described, and signaling mechanisms have been elucidated. Here, we review recent findings and interesting breakthroughs in bacterial TCS research. Furthermore, we discuss structural features, mechanisms of activation and regulation, and cross-regulation of RRs, with a focus on the largest RR family, OmpR/PhoB, to provide a comprehensive overview of these critically important signaling molecules.

show abstract

Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

Silversmith¹,

Bourret²

2018

Methods in Molecular Biology

View full text Add to dashboard Cite

The Escherichia coli chemotaxis protein CheY is a model receiver domain containing a native tryptophan residue that serves as a fluorescent probe for CheY autophosphorylation with small molecule phosphodonors. Here we describe fluorescence measurement of apparent bimolecular rate constants for reaction of wild type and mutant CheY with phosphodonors acetyl phosphate, phosphoramidate, or monophosphoimidazole. Step-by-step protocols to synthesize phosphoramidate (K salt) and monophosphoimidazole (Na salt), which are not commercially available, are provided. Key factors to consider in developing autophosphorylation assays for other response regulators are also discussed.

show abstract

A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB

Cited by 40 publications

References 57 publications

Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics

Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics

Prokaryotic 2-component systems and the OmpR/PhoB superfamily

Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

Contact Info

Product

Resources

About