Structural transitions in the FixJ receiver domain

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.     

Dimer-induced signal propagation in Spo0A

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.     

Evidence for phosphorylation-dependent conformational changes in methylesterase CheB

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.     

Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101

FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti. Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation. These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations. The path for the conformational change was studied with a distance constraint directing the system from one state to the other. The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101. A model presenting the sequence of events during the activation/deactivation process is discussed.     

High-resolution solution structure of the beryllofluoride-activated NtrC receiver domain

Bacterial receiver domains mediate the cellular response to environmental changes through conformational changes induced by phosphorylation of a conserved aspartate residue. While the structures of several activated receiver domains have recently been determined, there is substantial variation in the conformational changes occurring upon activation. Here we present the high-resolution structure of the activated NtrC receiver domain (BeF(3)(-)-NtrC(r) complex) determined using NMR data, including residual dipolar couplings, yielding a family of structures with a backbone rmsd of 0.57 +/- 0.08 A, which is compared with the previous lower-resolution structure of the phosphorylated protein. Both phosphorylation and beryllofluoride addition induce a shift in register and an axial rotation of alpha-helix 4. In this high-resolution structure, we are able to observe a concerted change in the positions of Thr82 and Tyr101; this correlated change in two conserved residues (termed Y-T coupling) has been considered a general feature of the conformational change in receiver domains upon activation. In NtrC, this correlated side chain shift, leading to the helix reorientation, is distinctly different from the smaller reorganization seen in other activated receiver domains, and involves numerous other residues which do not participate in conformational changes seen in the other systems. Titration of the activated receiver domain with peptides from the NtrC ATPase domain provides direct evidence for interactions on the rearranged face of the receiver domain, which are likely to be responsible for enabling assembly into the active aggregate. Analysis of the active structure also suggests that His84 may play a role in controlling the phosphate hydrolysis rate.     

Crystal structure of a cyanobacterial phytochrome response regulator

The two-component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade. Rcp1 is the phosphate receiver module in a two-component system controlling the light response of cyanobacteria Synechocystis sp. via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light. Here we describe the crystal structure of Rcp1 refined to a crystallographic R-factor of 18.8% at a resolution of 1.9 A. The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five-stranded parallel beta-sheets forming a single-domain protein homologous with the N-terminal activator domain of other response regulators (e.g., chemotaxis protein CheY). The three-dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C-terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface. The involvement of the residues undergoing phosphorylation-induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.     

The Residue Threonine 82 of DevR (DosR) Is Essential for DevR Activation and Function in Mycobacterium tuberculosis Despite Its Atypical Location

The DevR (DosR) response regulator initiates the bacterial adaptive response to a variety of signals, including hypoxia in in vitro models of dormancy. Its receiver domain works as a phosphorylation-mediated switch to activate the DNA binding property of its output domain. Receiver domains are characterized by the presence of several highly conserved residues, and these sequence features correlate with structure and hence function. In response regulators, interaction of phosphorylated aspartic acid at the active site with the conserved threonine is believed to be crucial for phosphorylation-mediated conformational change. DevR contains all the conserved residues, but the structure of its receiver domain in the unphosphorylated protein is strikingly different, and key threonine (T82), tyrosine (Y101), and lysine (K104) residues are placed uncharacteristically far from the D54 phosphorylation site. In view of the atypical location of T82 in DevR, the present study aimed to examine the importance of this residue in the activation mechanism. Mycobacterium tuberculosis expressing a DevR T82A mutant protein is defective in autoregulation and supports hypoxic induction of the DevR regulon only very weakly. These defects are ascribed to slow and partial phosphorylation and the failure of T82A mutant protein to bind cooperatively with DNA. Our results indicate that the T82 residue is crucial in implementing conformational changes in DevR that are essential for cooperative binding and for subsequent gene activation. We propose that the function of the T82 residue in the activation mechanism of DevR is conserved in spite of the unusual architecture of its receiver domain.     

Multistate allostery in response regulators: phosphorylation and mutagenesis activate RegA via alternate modes

Response regulators (RRs) belong to two-component signaling pathways, widely prevalent in bacteria and lower eukaryotes, for sensing and mediating responses to diverse environmental stress stimuli. RRs are modular proteins, and in most instances, a receiver domain is found connected to diverse effector domain(s). All receiver domains contain a conserved aspartate, which is the site of phosphorylation by an associated histidine kinase. RRs function as phosphorylatable signaling switches whereby histidine-kinase-mediated phosphorylation of RRs alters its output function. It is largely unknown how phosphorylation of the receiver domain triggers activation of distally positioned effector domain(s). Although crystal structures have highlighted differences in conformations from comparisons of snapshots of the unphosphorylated and phosphorylated receiver domains, how this is translated into altered activity of a distal effector domain has remained a mystery. While allosteric relays have been identified within receiver domains by NMR and X-ray crystallography, phosphorylated states of larger multidomain RRs have not yet been characterized. In this study, we have used amide hydrogen/deuterium exchange mass spectrometry to probe the conformational dynamics of a multidomain RR, RegA from Dictyostelium discoideum, by comparisons of the unphosphorylated and phosphorylated states and an activating mutant. Our results reveal allosteric coupling between the site of phosphorylation and the activating mutation. Interestingly, however, the conformations of the effector domains in both instances are distinct. Hydrogen/deuterium exchange mass spectrometry indicates that the 'inactive' and 'active' conformations exist as ensembles of multiple conformations. This is consistent with the 'conformational selection' model for describing phosphorylation-dependent regulation of multidomain RRs.     

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

Two‐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.     

Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis.

In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV. CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain. In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P). This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB. CheV-P is also highly stable in comparison with CheB-P. Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein. Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine. In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine. Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins. Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW, this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant. A possible structural basis for this feature is discussed.     

Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages

whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although bldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for bldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. bldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.