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.

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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Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the β5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheA P1 , suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.

BackgroundMany bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners.ResultsWe report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1.ConclusionsPhosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.Electronic supplementary materialThe online version of this article (10.1186/s12858-019-0104-5) contains supplementary material, which is available to authorized users.

Rapid response to external stimuli is crucial for survival and proliferation of microorganisms. Pathogenic fungi employ histidine-to-aspartate multistep phosphorelay systems to respond to environmental stress, progress through developmental stages and to produce virulence factors. Because these His-to-Asp phosphorelay systems are not found in humans, they are potential targets for the development of new antifungal therapies. Here we report the characterization of the histidine phosphotransfer (HPt) protein Ypd1 from the human fungal pathogen Cryptococcus neoformans. Results from this study demonstrate that CnYpd1 indeed functions as a phosphorelay protein in vitro, and that H138 is confirmed as the site of phosphorylation. We found that CnYpd1 exhibits unique characteristics in comparison to other histidine phosphotransfer proteins, such as an extended N-terminal amino acid sequence, which we find contributes to structural integrity, a longer phosphorylated life time and the ability to bind calcium ions.

The rapid increase of ‘-omics' data warrants the reconsideration of experimental strategies to investigate general protein function. Studying individual members of a protein family is likely insufficient to provide a complete mechanistic understanding of family functions, especially for diverse families with thousands of known members. Strategies that exploit large amounts of available amino acid sequence data can inspire and guide biochemical experiments, generating broadly applicable insights into a given family. Here we review several methods that utilize abundant sequence data to focus experimental efforts and identify features truly representative of a protein family or domain. First, coevolutionary relationships between residues within primary sequences can be successfully exploited to identify structurally and/or functionally important positions for experimental investigation. Second, functionally important variable residue positions typically occupy a limited sequence space, a property useful for guiding biochemical characterization of the effects of the most physiologically and evolutionarily relevant amino acids. Third, amino acid sequence variation within domains shared between different protein families can be used to sort a particular domain into multiple subtypes, inspiring further experimental designs. Although generally applicable to any kind of protein domain because they depend solely on amino acid sequences, the second and third approaches are reviewed in detail because they appear to have been used infrequently and offer immediate opportunities for new advances. Finally, we speculate that future technologies capable of analyzing and manipulating conserved and variable aspects of the three-dimensional structures of a protein family could lead to broad insights not attainable by current methods.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against the pertussis toxin but does not prevent transmission or colonization. Cases of B. pertussis infections are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past three days in the lung, suggesting PlrSR works in a phosphorylation dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 and Glu522 were essential for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS can both phosphotransfer to and exert phosphatase activity towards PlrR. Unexpectedly, PlrR forms a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.