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

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p>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 <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> 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 BeF<jats:sub>3</jats:sub><jats:sup>−</jats:sup> are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> 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.</jats:p>

Predicted Functional and Structural Diversity of Receiver Domains in Fungal Two-Component Regulatory Systems

“…Six variable positions are known to affect the rates of phosphorylation and dephosphorylation reactions in bacterial receiver domains. Five of these appear to be functionally linked, as revealed by sequence covariation ( 19 ). T+1 directly controls physical access to the phosphorylation site ( 13 , 20 ).…”

“…D+2 and T+2 surround the path to the phosphorylation site and exert influence via hydrophobic/hydrophilic interactions with the reactants and products ( 18 , 21 , 22 ). K+1, K+2, and K+4 affect the equilibria between catalytically active and inactive conformations ( 19 , 23 ). Different types of bacterial response regulators exhibit different combinations of amino acids at these variable positions, consistent with multivariate evolutionary tuning of reaction kinetics to suit their biological processes.…”

“…Five types of fungal HHK receiver domains contain Phe residues or functionally similar Tyr residues at K+2. However, 15 types of fungal HHK receiver domains contain primarily an Ile, Leu, or Val residue at K+2, which is likely to increase the fraction of the population in active conformations and enhance phosphorylation ( 19 ). K–3 (involved in allosteric communication between the active site and the α4β5α5 surface) is mostly Tyr or Phe residues in bacteria, as well as in most types of fungal HHK receiver domains.…”

“…Five types of fungal HHK receiver domains contain Phe residues or functionally similar Tyr residues at K+2. However, 15 types of fungal HHK receiver domains contain primarily an Ile, Leu, or Val residue at K+2, which is likely to increase the fraction of the population in active conformations and enhance phosphorylation ( 19 ).…”

Phospho-dependent Signaling during the General Stress Response by the Atypical Response Regulator and ClpXP Adaptor RssB

“…The K+1 residue, Pro 109 , is invariant, while K+2 (residue 110) is conserved as a branched amino acid (Supplemental Figure 1). These two residues also affect the kinetics of REC phosphorylation/dephosphorylation, but unlike residue D+2, they likely do so via an indirect mechanism that affects REC conformational equilibria 27 . Consistent with this, Val 110 is located about 12Å away from the phosphorylation site and does not engage in any hydrogen bonds or van der Waals contacts that could link it directly to the active site.…”