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

2020 ◽  
Vol 202 (15) ◽  
Author(s):  
Philip B. Straughn ◽  
Luke R. Vass ◽  
Chase Yuan ◽  
Emily N. Kennedy ◽  
Clay A. Foster ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reaction Kinetics ◽  
Rate Constants ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Signal Propagation ◽  
Reaction Rates ◽  
Phosphoryl Group ◽  
Response Regulators ◽  
Phosphoryl Groups

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.

scholarly journals Investigation of the Role of Electrostatic Charge in Activation of the Escherichia coli Response Regulator CheY

2003 ◽  
Vol 185 (21) ◽  
pp. 6385-6391 ◽  
Author(s):  
Jenny G. Smith ◽  
Jamie A. Latiolais ◽  
Gerald P. Guanga ◽  
Sindhura Citineni ◽  
Ruth E. Silversmith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Amino Acid ◽  
Active Site ◽  
Response Regulator ◽  
Residual Activity ◽  
Phosphoryl Group ◽  
Sensor Kinase ◽  
Amino Acid Substitutions ◽  
Flagellar Rotation

ABSTRACT In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg2+ ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg2+-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.

scholarly journals Identification of Z nucleotides as an ancient signal for two-component system activation in bacteria

2020 ◽  
Vol 117 (52) ◽  
pp. 33530-33539
Author(s):  
Oscar J. Vázquez-Ciros ◽  
Adrián F. Alvarez ◽  
Dimitris Georgellis
Keyword(s):  
De Novo ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Response Regulators ◽  
Carbamoyl Phosphate ◽  
Cellular Processes ◽  
Wide Range ◽  
Two Component ◽  
Two Component Systems

Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.

scholarly journals Dynamics and activation in response regulators: the β4-α4 loop

2012 ◽  
Vol 3 (2) ◽  
pp. 175-182 ◽  
Author(s):  
Benjamin G. Bobay ◽  
James A. Hoch ◽  
John Cavanagh
Keyword(s):  
Response Regulator ◽  
Short Review ◽  
Phosphoryl Group ◽  
Sensor Kinase ◽  
Response Regulators ◽  
Component Systems ◽  
Two Component ◽  
Phosphorylation Event ◽  
Primary Means ◽  
Two Component Systems

AbstractTwo-component signal transduction systems of microbes are a primary means to respond to signals emanating from environmental and metabolic fluctuations as well as to signals coordinating the cell cycle with macromolecular syntheses, among a large variety of other essential roles. Signals are recognized by a sensor domain of a histidine kinase which serves to convert signal binding to an active transmissible phosphoryl group through a signal-induced ATP-dependent autophosphorylation reaction directed to histidine residue. The sensor kinase is specifically mated to a response regulator, to which it transfers the phosphoryl group that activates the response regulator’s function, most commonly gene repression or activation but also interaction with other regulatory proteins. Two-component systems have been genetically amplified to control a wide variety of cellular processes; for example, both Escherichia coli and Pseudomonas aeruginosa have 60 plus confirmed and putative two-component systems. Bacillus subtilis has 30 plus and Nostoc punctiformis over 100. As genetic amplification does not result in changes in the basic structural folds of the catalytic domains of the sensor kinase or response regulators, each sensor kinase must recognize its partner through subtle changes in residues at the interaction surface between the two proteins. Additionally, the response regulator must prepare itself for efficient activation by the phosphorylation event. In this short review, we discuss the contributions of the critical β4-α4 recognition loop in response regulators to their function. In particular, we focus on this region’s microsecond-millisecond timescale dynamics propensities and discuss how these motions play a major role in response regulator recognition and activation.

scholarly journals Chemotactic Signaling by an Escherichia coli CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners

2004 ◽  
Vol 186 (9) ◽  
pp. 2664-2672 ◽  
Author(s):  
Knut Jahreis ◽  
Tom B. Morrison ◽  
Andrés Garzón ◽  
John S. Parkinson
Keyword(s):  
Escherichia Coli ◽  
Phosphorylation Site ◽  
Binding Domain ◽  
Response Regulators ◽  
Signaling Specificity ◽  
Phosphoryl Groups ◽  
Turn Over ◽  
Chemotactic Ability

ABSTRACT CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheAΔP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheAΔP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheAΔP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheAΔP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.

scholarly journals Amino acid identity at one position within the α1 helix of both the histidine kinase and the response regulator of the WalRK and PhoPR two-component systems plays a crucial role in the specificity of phosphotransfer

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1848-1859 ◽  
Author(s):  
Inga Jende ◽  
Kottayil I. Varughese ◽  
Kevin M. Devine
Keyword(s):  
Amino Acid ◽  
Response Regulator ◽  
Amino Acid Identity ◽  
Response Regulators ◽  
Acid Identity ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Interacting Surfaces ◽  
High Level

Two-component systems usually function as cognate pairs, thereby ensuring an appropriate response to the detected signal. The ability to exclusively phosphorylate a partner protein, often in the presence of many competing homologous substrates, demonstrates a high level of specificity that must derive from the interacting surfaces of the two-component system. Here, we identify positions within the histidine kinases and response regulators of the WalRK and PhoPR two-component systems of Bacillus subtilis that make a major contribution to the specificity of phosphotransfer. Changing the identity of the amino acid at position 11 within the α1 helix of WalK and at position 17 within the α1 helix of PhoP altered discrimination and allowed phosphotransfer to occur with the non-cognate partner. Changing amino acids at additional positions of the WalK kinase increased phosphotransfer, while changes at additional positions in PhoP only had an effect in the presence of the change at position 17. The importance of amino acid identity at these two positions is supported by the fact that the amino acid combinations of Ile and Ser in WalRK, and Leu and Gly in PhoPR, are very highly conserved among orthologues, while modelling indicates that these amino acid pairs are juxtaposed in the WalRK and PhoPR complexes.

scholarly journals Ssk1p Response Regulator Binding Surface on Histidine- Containing Phosphotransfer Protein Ypd1p

2003 ◽  
Vol 2 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Stace W. Porter ◽  
Qingping Xu ◽  
Ann H. West
Keyword(s):  
Response Regulator ◽  
Signal Transduction Pathway ◽  
Phosphoryl Group ◽  
Response Regulators ◽  
Alanine Scanning Mutagenesis ◽  
Hydrophobic Residues ◽  
Strength Of Interaction ◽  
Binding Surface ◽  
Terminal Response ◽  
Membrane Bound

ABSTRACT Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the αA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.

Kinetic Solvent Effects in Organic Reactions

2017 ◽  
Author(s):  
Belinda Slakman ◽  
Richard West
Keyword(s):  
Solvent Effect ◽  
Reaction Kinetics ◽  
Computational Methods ◽  
High Throughput ◽  
Kinetic Modeling ◽  
Solvent Effects ◽  
Reaction Rates ◽  
Prior Work ◽  
Solvent Phase ◽  
High Throughput Manner

<div> <div> <div> <p>This article reviews prior work studying reaction kinetics in solution, with the goal of using this information to improve detailed kinetic modeling in the solvent phase. Both experimental and computational methods for calculating reaction rates in liquids are reviewed. Previous studies, which used such methods to determine solvent effects, are then analyzed based on reaction family. Many of these studies correlate kinetic solvent effect with one or more solvent parameters or properties of reacting species, but it is not always possible, and investigations are usually done on too few reactions and solvents to truly generalize. From these studies, we present suggestions on how best to use data to generalize solvent effects for many different reaction types in a high throughput manner. </p> </div> </div> </div>

Kinetics of esterification of monoisopropylphenols with phosphoryl trichloride

1989 ◽  
Vol 54 (5) ◽  
pp. 1311-1317
Author(s):  
Miroslav Magura ◽  
Ján Vojtko ◽  
Ján Ilavský
Keyword(s):  
Liquid Phase ◽  
Rate Constants ◽  
Reaction Rates ◽  
Activation Energies ◽  
The Individual ◽  
Kinetics Of

The kinetics of liquid-phase isothermal esterification of POCl3 with 2-isopropylphenol and 4-isopropylphenol have been studied within the temperature intervals of 110 to 130 and 90 to 110 °C, respectively. The rate constants and activation energies of the individual steps of this three-step reaction have been calculated from the values measured. The reaction rates of the two isomers markedly differ: at 110 °C 4-isopropylphenol reacts faster by the factors of about 7 and 20 for k1 and k3, respectively. This finding can be utilized in preparation of mixed triaryl phosphates, since the alkylation mixture after reaction of phenol with propene contains an excess of 2-isopropylphenol over 4-isopropylphenol.

scholarly journals FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wei Liu ◽  
Xue Bai ◽  
Yan Li ◽  
Haikun Zhang ◽  
Xiaoke Hu
Keyword(s):  
Signal Transduction ◽  
Host Plant ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Response Regulators ◽  
Free Living ◽  
Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

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