Structure of McsB, a protein kinase for regulated arginine phosphorylation.
ABSTRACT: Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.
Project description:Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) has impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate amidines and sulfonate amidines as isosteres of pArg and then use these mimics as haptens to develop the first high-affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation.
Project description:Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.
Project description:Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC-ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC-ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC-ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system.
Project description:Arginine phosphorylation is an emerging post-translational protein modification implicated in the bacterial stress response. Although early reports suggested that arginine phosphorylation also occurs in higher eukaryotes, its overall prevalence was never studied using modern mass spectrometry methods, owing to technical difficulties arising from the acid lability of phosphoarginine. As shown recently, the McsB and YwlE proteins from Bacillus subtilis function as a highly specific protein arginine kinase and phosphatase couple, shaping the phosphoarginine proteome. Using a B. subtilis ?ywlE strain as a source for arginine-phosphorylated proteins, we were able to adapt mass spectrometry (MS) protocols to the special chemical properties of the arginine modification. Despite this progress, the analysis of protein arginine phosphorylation in eukaryotes is still challenging, given the great abundance of serine/threonine phosphorylations that would compete with phosphoarginine during the phosphopeptide enrichment procedure, as well as during data-dependent MS acquisition. We thus set out to establish a method for the selective enrichment of arginine-phosphorylated proteins as an initial step in the phosphoproteomic analysis. For this purpose, we developed a substrate-trapping mutant of the YwlE phosphatase that retains binding affinity toward arginine-phosphorylated proteins but cannot hydrolyze the captured substrates. By testing a number of active site substitutions, we identified a YwlE mutant (C9A) that stably binds to arginine-phosphorylated proteins. We further improved the substrate-trapping efficiency by impeding the oligomerization of the phosphatase mutant. The engineered YwlE trap efficiently captured arginine-phosphorylated proteins from complex B. subtilis ?ywlE cell extracts, thus facilitating identification of phosphoarginine sites in the large pool of cellular protein modifications. In conclusion, we present a novel tool for the selective enrichment and subsequent MS analysis of arginine phosphorylation, which is a largely overlooked protein modification that might be important for eukaryotic cell signaling.
Project description:The soil bacterium Bacillus subtilis possesses a fine-tuned and complex heat stress response system. The repressor CtsR, whose activity is regulated by its modulators McsA and McsB, controls the expression of the cellular protein quality control genes clpC, clpE and clpP. Here, we show that the interaction of McsA and McsB with CtsR results in the formation of a ternary complex that not only prevents the binding of CtsR to its target DNA, but also results in a subsequent phosphorylation of McsB, McsA and CtsR. We further demonstrate that McsB is a tyrosine kinase that needs McsA to become activated. ClpC inhibits the kinase activity of McsB, indicating a direct role in initiating CtsR-controlled heat shock response. Interestingly, the kinase domain of McsB is homologous to guanidino phosphotransferase domains originating from eukaryotic arginine and creatine kinases. Mutational analysis of key residues of the guanidino kinase domain demonstrated that McsB utilizes this domain to catalyze the tyrosine phosphorylation. McsB represents therefore a new kind of tyrosine kinase, driven by a guanidino phosphotransferase domain.
Project description:In Bacillus subtilis, the Spx transcription factor controls a large regulon in response to disulfide, heat, and cell wall stresses. The regulatory mechanisms that activate the Spx regulon are remarkably complex and involve changes in transcription, proteolysis, and posttranslational modifications. To identify genes involved in Spx regulation, we performed a transposon screen for mutations affecting expression of trxB, an Spx-dependent gene. Inactivation of ctsR, encoding the regulator of the Clp proteases, reduced trxB expression and lowered Spx levels. This effect required ClpP, but involved ClpC rather than the ClpX unfoldase. Moreover, cells lacking McsB, a dual function arginine kinase and ClpCP adaptor, largely reverted the ctsR phenotype and increased trxB expression. The role of McsB appears to involve its kinase activity, since loss of the YwlE phosphoarginine phosphatase also led to reduced trxB expression. Finally, we show that Spx is itself a regulator of the ctsR operon. Altogether, this work provides evidence for a role of CtsR regulon members ClpC, ClpP, and McsB in Spx regulation and identifies a new feedback pathway associated with Spx activity in B. subtilis IMPORTANCE In Bacillus subtilis, the Spx transcription factor is proteolytically unstable, and protein stabilization figures prominently in the induction of the Spx regulon in response to oxidative and cell envelope stresses. ClpXP is largely, but not entirely, responsible for Spx instability. Here, we identify ClpCP as the protease that degrades Spx under conditions that antagonize the ClpXP pathway. Spx itself contributes to activation of the ctsR operon, which encodes ClpC as well as the McsB arginine kinase and protease adaptor, thereby providing a negative feedback mechanism. Genetic studies reveal that dysregulation of the CtsR regulon or inactivation of the YwlE phosphoarginine phosphatase decreases Spx activity through mechanisms involving both protein degradation and posttranslational modification.
Project description:Ever since the identification of the first protein-arginine kinase, McsB, in B. subtilis arginine phosphorylation gained more attention. However, the analysis of phosphorylations especially phosphormaidates comes along with several challenges. The sub-stoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis and the acid instability of phosphoramidates was long though to impede those enrichments. Further good spectral quality is required which can be reduced by the presence of neutral losses of phosphoric acid upon higher energy collision induced dissociation. Adaptation of common enrichment strategies to less acidic conditions and the use of electron-transfer dissociation allowed the identification of more than 200 pArg sites in B. subtilis and S. aureus so far. Here we show that pArg is stable enough for commonly used Fe3+-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for phosphoproteomics. By profiling a serine/ threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic phosphoarginine peptides we validated the presence and localization of arginine phosphorylation in S. aureus and lastly, we could show that Stp1 is influencing the arginine phosphoproteom without being a protein-arginine phosphatase.
Project description:Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase ?B regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.
Project description:Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and l-arginine and is a monomeric homolog of the human enzyme creatine kinase. Arginine and creatine kinases belongs to the phosphagen kinase family of enzymes, which consists of eight known members, each of which is specific for its own phosphagen. Here, the source of phosphagen specificity in arginine kinase is investigated through the use of phosphagen analogs. Crystal structures have been determined for Limulus polyphemus arginine kinase with one of four arginine analogs bound in a transition state analog complex: l-ornithine, l-citrulline, imino-l-ornithine, and d-arginine. In all complexes, the enzyme achieves a closed conformation very similar to that of the cognate transition state analog complex, but differences are observed in the configurations of bound ligands. Arginine kinase exhibits no detectable activity towards ornithine, citrulline, or imino-l-ornithine, and only trace activity towards d-arginine. The crystal structures presented here demonstrate that phosphagen specificity is derived neither from a lock-and-key mechanism nor a modulation of induced-fit conformational changes, but potentially from subtle distortions in bound substrate configurations.
Project description:Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.