Phosphorylation Of Receiver Domain Research Articles

Evolutionary diversification of the RomR protein of the invasive deltaproteobacterium, Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a predatory deltaproteobacterium that encounters individual Gram-negative prey bacteria with gliding or swimming motility, and then is able to invade such prey cells via type IVa pilus-dependent mechanisms. Movement control (pili or gliding) in other deltaproteobacteria, such as the pack hunting Myxococcus xanthus, uses a response regulator protein, RomRMx (which dynamically relocalises between the cell poles) and a GTPase, MglAMx, previously postulated as an interface between the FrzMx chemosensory system and gliding or pilus-motility apparatus, to produce regulated bidirectional motility. In contrast, B. bacteriovorus predation is a more singular encounter between a lone predator and prey; contact is always via the piliated, non-flagellar pole of the predator, involving MglABd, but no Frz system. In this new study, tracking fluorescent RomRBd microscopically during predatory growth shows that it does not dynamically relocalise, in contrast to the M. xanthus protein; instead having possible roles in growth events. Furthermore, transcriptional start analysis, site-directed mutagenesis and bacterial two-hybrid interaction studies, indicate an evolutionary loss of RomRBd activation (via receiver domain phosphorylation) in this lone hunting bacterium, demonstrating divergence from its bipolar role in motility in pack-hunting M. xanthus and further evolution that may differentiate lone from pack predators.

Use of restrained molecular dynamics to predict the conformations of phosphorylated receiver domains in two-component signaling systems.

ABSTRACTTwo‐component signaling (TCS) is the primary means by which bacteria, as well as certain plants and fungi, respond to external stimuli. Signal transduction involves stimulus‐dependent autophosphorylation of a sensor histidine kinase and phosphoryl transfer to the receiver domain of a downstream response regulator. Phosphorylation acts as an allosteric switch, inducing structural and functional changes in the pathway's components. Due to their transient nature, phosphorylated receiver domains are challenging to characterize structurally. In this work, we provide a methodology for simulating receiver domain phosphorylation to predict conformations that are nearly identical to experimental structures. Using restrained molecular dynamics, phosphorylated conformations of receiver domains can be reliably sampled on nanosecond timescales. These simulations also provide data on conformational dynamics that can be used to identify regions of functional significance related to phosphorylation. We first validated this approach on several well‐characterized receiver domains and then used it to compare the upstream and downstream components of the fungal Sln1 phosphorelay. Our results demonstrate that this technique provides structural insight, obtained in the absence of crystallographic or NMR information, regarding phosphorylation‐induced conformational changes in receiver domains that regulate the output of their associated signaling pathway. To our knowledge, this is the first time such a protocol has been described that can be broadly applied to TCS proteins for predictive purposes. Proteins 2016; 85:155–176. © 2016 Wiley Periodicals, Inc.

Structure, Regulation, and Inhibition of the Quorum-Sensing Signal Integrator LuxO

In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors’ kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.

Cross Talk Inhibition Nullified by a Receiver Domain Missense Substitution.

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. Many 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.

The structural basis of the response regulator DrRRA from Deinococcus radiodurans

DrRRA, a vital and recently discovered gene product of Deinococcus radiodurans, is a member of the OmpR/PhoB family of response regulators that couple with the cognate histidine kinase (HK) to form a typical two component system (TCS). It is known that the DrRRA is responsible for the transcriptional levels of numerous genes mostly relating to the stress response and DNA repair. In this paper, the crystal structures of the effector domain and full-length protein of DrRRA with resolutions of 1.6 and 2.3Å, respectively, are determined. These crystal structures depicted that DrRRA has the structural features of the OmpR/PhoB subfamily and were also confirmed by SAXS investigation of the protein in solution. Our data suggest that the receiver domain blocks the binding of DNA to the DNA recognition helix of effector domain; while the interdomain interface would be unwrapped, via the phosphorylation of receiver domain and/or the inducement of DNA, in order to provide DNA binding.

An atypical receiver domain controls the dynamic polar localization of the Myxococcus xanthus social motility protein FrzS

The Myxococcus xanthus FrzS protein transits from pole-to-pole within the cell, accumulating at the pole that defines the direction of movement in social (S) motility. Here we show using atomic-resolution crystallography and NMR that the FrzS receiver domain (RD) displays the conserved switch Tyr102 in an unusual conformation, lacks the conserved Asp phosphorylation site, and fails to bind Mg2+ or the phosphoryl analogue, Mg2+·BeF3. Mutation of Asp55, closest to the canonical site of RD phosphorylation, showed no motility phenotype in vivo, demonstrating that phosphorylation at this site is not necessary for domain function. In contrast, the Tyr102Ala and His92Phe substitutions on the canonical output face of the FrzS RD abolished S-motility in vivo. Single-cell fluorescence microscopy measurements revealed a striking mislocalization of these mutant FrzS proteins to the trailing cell pole in vivo. The crystal structures of the mutants suggested that the observed conformation of Tyr102 in the wild-type FrzS RD is not sufficient for function. These results support the model that FrzS contains a novel ‘pseudo-receiver domain’ whose function requires recognition of the RD output face but not Asp phosphorylation.

The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p.

The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.

Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.