Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

Silversmith, Ruth E.; Bourret, Robert B.

doi:10.1007/978-1-4939-7577-8_25

Cited by 3 publications

(3 citation statements)

References 47 publications

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“…To determine the autophosphorylation rate constant, k phos /K S , we used a new method (see Materials and Methods). The traditional method measures the observed pre-steady-state rate constant, k obs , for approach to equilibrium between autophosphorylation and autodephosphorylation reactions (16,40,50). In contrast, the new method is based on a steady-state measurement and involves determination of K 1/2 , the concentration of phosphodonor required to phosphorylate half of the CheY population (51).…”

Section: Resultsmentioning

confidence: 99%

“…Determination of K 1/2 . The small-molecule phosphodonor potassium phosphoramidate was synthesized as described previously (50,88). The purity of phosphoramidate was determined by 31 P nuclear magnetic resonance (NMR).…”

Section: Discussionmentioning

confidence: 99%

“…Determination of k phos /K S . The rate of approach to equilibrium between autophosphorylation and autophosphorylation k obs was determined at multiple phosphoramidate concentrations as previously described (50). The autophosphorylation rate constant, k phos /K S , is the slope of the best-fit line of a plot of k obs versus the phosphoramidate concentration.…”

Section: Discussionmentioning

confidence: 99%

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Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation

et al. 2020

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ABSTRACT Microorganisms and plants utilize two-component systems to regulate adaptive responses to changing environmental conditions. Sensor kinases detect stimuli and alter their autophosphorylation activity accordingly. Signal propagation occurs via the transfer of phosphoryl groups from upstream kinases to downstream response regulator proteins. Removal of phosphoryl groups from the response regulator typically resets the system. Members of the same protein family may catalyze phosphorylation and dephosphorylation reactions with different efficiencies, exhibiting rate constants spanning many orders of magnitude to accommodate response time scales from milliseconds to days. We previously found that variable positions one or two residues to the C-terminal side of the conserved Asp phosphorylation site (D+2) or Thr/Ser (T+1/T+2) in response regulators alter reaction kinetics by direct interaction with phosphodonor or phosphoacceptor molecules. Here, we explore the kinetic effects of amino acid substitutions at the two positions immediately C-terminal to the conserved Lys (K+1/K+2) in the model Escherichia coli response regulator CheY. We measured CheY autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues that represent 84% of naturally occurring response regulators. Effects on autodephosphorylation were modest, but autophosphorylation rate constants varied by 2 orders of magnitude, suggesting that the K+1/K+2 positions influence reaction kinetics by altering the conformational spectrum sampled by CheY at equilibrium. Additional evidence supporting this indirect mechanism includes the following: the effect on autophosphorylation rate constants is independent of the phosphodonor, the autophosphorylation rate constants and dissociation constants for the phosphoryl group analog BeF3− are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site. IMPORTANCE We have identified five variable positions in response regulators that allow the rate constants of autophosphorylation and autodephosporylation reactions each to be altered over 3 orders of magnitude in CheY. The distributions of variable residue combinations across response regulator subfamilies suggest that distinct mechanisms associated with different variable positions allow reaction rates to be tuned independently during evolution for diverse biological purposes. This knowledge could be used in synthetic-biology applications to adjust the properties (e.g., background noise and response duration) of biosensors and may allow prediction of response regulator reaction kinetics from the primary amino acid sequence.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation

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Measuring the Activities of Two-Component Regulatory System Phosphatases

Bourret

Silversmith

2018

Methods in Enzymology

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Azorhizobium caulinodans Chemotaxis Is Controlled by an Unusual Phosphorelay Network

et al. 2022

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Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata . This agriculturally significant symbiotic relationship is important in lowland rice cultivation, and allows for nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA , cheW , cheY2 , cheB , and cheR . Two other chemotaxis genes, cheY1 and cheZ , are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/Receiver pair (termed W2-Rec), follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 or W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1, but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move towards nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over non-specific motion. CheY is an essential mediator of the chemotactic response with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.

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Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

Cited by 3 publications

References 47 publications

Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation

Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation

Measuring the Activities of Two-Component Regulatory System Phosphatases

Azorhizobium caulinodans Chemotaxis Is Controlled by an Unusual Phosphorelay Network

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