Cross Talk Inhibition Nullified by a Receiver Domain Missense Substitution

Abstract

In two-component signal transduction, a sensor protein transmitter module controls cognate receiver domain phosphorylation. Most receiver domain sequences contain a small residue (Gly or Ala) at position T ؉ 1 just distal to the essential Thr or Ser residue that forms part of the active site. However, some members of the NarL receiver subfamily have a large hydrophobic residue at position T ؉ 1. Our laboratory previously isolated a NarL mutant in which the T ؉ 1 residue Val-88 was replaced with an orthodox small Ala. This NarL V88A mutant confers a striking phenotype in which high-level target operon expression is both signal (nitrate) and sensor (NarX and NarQ) independent. This suggests that the NarL V88A protein is phosphorylated by cross talk from noncognate sources. Although cross talk was enhanced in ackA null strains that accumulate acetyl phosphate, it persisted in pta ackA double null strains that cannot synthesize this compound and was observed also in narL ؉ strains. This indicates that acetate metabolism has complex roles in mediating NarL cross talk. Contrariwise, cross talk was sharply diminished in an arcB barA double null strain, suggesting that the encoded sensors contribute substantially to NarL V88A cross talk. Separately, the V88A substitution altered the in vitro rates of NarL autodephosphorylation and transmitter-stimulated dephosphorylation and decreased affinity for the cognate sensor, NarX. Together, these experiments show that the residue at position T ؉ 1 can strongly influence two distinct aspects of receiver domain function, the autodephosphorylation rate and cross talk inhibition. IMPORTANCEMany bacterial species contain a dozen or more discrete sensor-response regulator two-component systems that convert a specific input into a distinct output pattern. Cross talk, the unwanted transfer of signals between circuits, occurs when a response regulator is phosphorylated inappropriately from a noncognate source. Cross talk is inhibited in part by the high interaction specificity between cognate sensor-response regulator pairs. This study shows that a relatively subtle missense change from Val to Ala nullifies cross talk inhibition, enabling at least two noncognate sensors to enforce an inappropriate output independently of the relevant input.T wo-component signal transduction determines numerous phenotypes in microorganisms. The sensor transmitter module, in response to the input signal, governs phosphorylation of the response regulator receiver domain to control the output (1). Although conserved in structure and sequence, receiver domains nevertheless catalyze autophosphorylation and autodephosphorylation over a broad range of rates (2) and interact specifically with their cognate transmitter modules (3).Receiver domains, approximately 120 residues, share a topology in which a central five-stranded parallel ␤-sheet is enveloped by five ␣-helices. The active site for phosphorylation and dephosphorylation includes five critical residues, as exemplified by the intensively studied C...

A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation

“…55 Residues that partially occlude the attacking water molecule, such as the β-branched amino acids Val (Figure 3B) and Ile, resulted in distinct decreases in autodephosphorylation rate constants in CheY and CheB (Tables 2 and 5). Conversely, removing the Val from wild type NarL enhanced the autodephosphorylation rate constant, in agreement with the findings of Huynh et al 69 Response Regulator Structural Activation May Precede Phosphodonor Binding. Response regulators undergo a structural rearrangement that facilitates phosphochemistry and switches the signaling state.…”

“…Conversely, removing the Val from wild type NarL enhanced the autodephosphorylation rate constant, in agreement with Huynh et al 69 …”

“…However, the phenotype of cells expressing NarLV88A does not depend on the small molecule phosphodonors acetyl phosphate or carbamoyl phosphate, and instead is due to reduced sensitivity to the phosphatase activity of NarX. 69 …”

“…For example, the addition of a single atom at position T+1 (the Ser hydroxyl oxygen of CheYA88S versus the Ala hydrogen of wild type CheY) was sufficient to change the interaction such that CheY no longer could accept a phosphoryl group from MBP-NarX 227 (Figure 6AB). Furthermore, there is precedence for modulation of signal fidelity by position T+1 for FixL/FixJ 68 and NarX/NarL 69 as described in the next section.…”

Characterization of the CrbS/R Two-Component System in Pseudomonas fluorescens Reveals a New Set of Genes under Its Control and a DNA Motif Required for CrbR-Mediated Transcriptional Activation

“…This suggests that CrbR is activated in E. coli through an unknown mechanism. Unspecific phosphorylation is a common occurrence in members of the OmpR/LuxR family (Barbieri et al, 2013 ; Huynh et al, 2015 ). The fact that the acs promoter is transcribed in E. coli is not surprising, considering that RpoD from Pseudomonas and its promoter consensus sequence are highly similar to those of E. coli (Potvin et al, 2008 ; Schulz et al, 2015 ).…”