Molecular basis of unidirectional information transmission in two-component systems: lessons from the DesK-DesR thermosensor

Cellular signaling systems transmit information over long distances using allosteric transitions and/or post-translational modifications. In two-component systems the sensor histidine kinase and response regulator are wired through phosphoryl-transfer reactions, using either a uni- or bi-directional transmission mode, allowing to build rich regulatory networks. Using the thermosensor DesK-DesR two-component system from Bacillus subtilis and combining crystal structures, QM/MM calculations and integrative kinetic modeling, we uncover that: i) longer or shorter distances between the phosphoryl-acceptor and -donor residues can shift the phosphoryl-transfer equilibrium; ii) the phosphorylation-dependent dimerization of the regulator acts as a sequestering mechanism by preventing the interaction with the histidine kinase; and iii) the kinase's intrinsic conformational equilibrium makes the phosphotransferase state unlikely in the absence of histidine phosphorylation, minimizing backwards transmission. These mechanisms allow the system to control the direction of signal transmission in a very efficient way, showcasing the key role that structure-encoded allostery plays in signaling proteins to store and transmit information.

PRD-Containing Virulence Regulators (PCVRs) in Pathogenic Bacteria

Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved pathway in bacteria that couples transport of sugars with phosphorylation to monitor host carbohydrate availability. A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Streptococcus pyogenes Mga and Bacillus anthracis AtxA. These paradigm PCVRs undergo phosphorylation, potentially via the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from Streptococcus pneumoniae (MgaSpn) and Enterococcus faecalis (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation via potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.

The many ways that nature has exploited the unusual structural and chemical properties of phosphohistidine for use in proteins

Histidine phosphorylation is an important and ubiquitous post-translational modification. Histidine undergoes phosphorylation on either of the nitrogens in its imidazole side chain, giving rise to 1- and 3- phosphohistidine (pHis) isomers, each having a phosphoramidate linkage that is labile at high temperatures and low pH, in contrast with stable phosphomonoester protein modifications. While all organisms routinely use pHis as an enzyme intermediate, prokaryotes, lower eukaryotes and plants also use it for signal transduction. However, research to uncover additional roles for pHis in higher eukaryotes is still at a nascent stage. Since the discovery of pHis in 1962, progress in this field has been relatively slow, in part due to a lack of the tools and techniques necessary to study this labile modification. However, in the past ten years the development of phosphoproteomic techniques to detect phosphohistidine (pHis), and methods to synthesize stable pHis analogues, which enabled the development of anti-phosphohistidine (pHis) antibodies, have accelerated our understanding. Recent studies that employed anti-pHis antibodies and other advanced techniques have contributed to a rapid expansion in our knowledge of histidine phosphorylation. In this review, we examine the varied roles of pHis-containing proteins from a chemical and structural perspective, and present an overview of recent developments in pHis proteomics and antibody development.

LHPP-Mediated Histidine Dephosphorylation Suppresses the Self-Renewal of Mouse Embryonic Stem Cells

Self-renewal of embryonic stem cells (ESCs) is orchestrated by a vast number of genes at the transcriptional and translational levels. However, the molecular mechanisms of post-translational regulatory factors in ESC self-renewal remain unclear. Histidine phosphorylation, also known as hidden phosphorylation, cannot be detected by conventional experimental methods. A recent study defined phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) as a histidine phosphatase, which regulates various biological behaviors in cells via histidine dephosphorylation. In this study, the doxycycline (DOX)-induced hLHPP-overexpressing mouse ESCs and mouse LHPP silenced mESCs were constructed. Quantitative polymerase chain reaction (qPCR), western blotting analysis, immunofluorescence, Flow cytometry, colony formation assays, alkaline phosphatase (AP) and bromodeoxyuridine (Brdu) staining were performed. We found that the histidine phosphorylation level was strikingly reduced following LHPP overexpression. Besides, the expression of Oct4 and Lefty1, indispensable genes in the process of ESCs self-renewal, was significantly down-regulated, while markers related to the differentiation were markedly elevated. Moreover, LHPP-mediated histidine dephosphorylation induced G0/G1 phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of Lhpp promoted the self-renewal of mESCs and reversed the RA induced increased expression of genes associated with differentiation. Mechanistically, our findings suggested that the enzymatic active site of LHPP was the cysteine residue at position 226, not 53. LHPP-mediated histidine dephosphorylation lowered the expression levels of β-catenin and the cell cycle-related genes CDK4 and CyclinD1, while it up-regulated the cell cycle suppressor genes P21 and P27. Taken together, our findings reveal that LHPP-mediated histidine dephosphorylation plays a role in the self-renewal of ESCs. LHPP-mediated histidine dephosphorylation inhibited the self-renewal of ESCs by negatively regulating the Wnt/β-catenin pathway and downstream cell cycle-related genes, providing a new perspective and regulatory target for ESCs self-renewal.

Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis–specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope–paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

31P NMR Spectroscopy Demonstrates Large Amounts of Phosphohistidine in Mammalian Cells

Protein phosphorylation plays a key role in many cellular processes but there is presently no accurate information or reliable procedure to determine the relative abundance of many phosphoamino acids in cells. At pH ≤ 8, phosphohistidine is unstable compared to the extensively studied phosphoserine, phosphothreonine and phosphotyrosine. This study reports the absolute quantitative analysis of histidine phosphorylation of proteins from a human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. The method was designed to minimize loss of the phosphohistidine phosphoryl group. Phosphohistidine was determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 20 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. After tryptic digest of proteins from the16HBE14o- cell lysate, the phosphohistidine signal was abolished and increasing phosphoserine/phosphothreonine signal was observed, which has implications for mass spectrometry investigations. The 31P NMR spectroscopic analysis not only highlights the abundance of phosphohistidine, which likely reflects its importance in mammalian cells, but also provides a way of measuring and comparing levels of phosphorylated amino acids in cells.

The phosphohistidine phosphatase SixA dephosphorylates the phosphocarrier NPr

Histidine phosphorylation is a post-translational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr, and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are out-competed by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The wide-spread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.

Colitis is associated with loss of LHPP and up-regulation of histidine phosphorylation in intestinal epithelial cells

Protein histidine phosphorylation (pHis) is a posttranslational modification involved in cell cycle regulation, ion channel activity and phagocytosis (1). Using novel monoclonal antibodies to detect pHis (2), we recently reported that loss of the histidine phosphatase LHPP results in elevated pHis levels in hepatocellular carcinoma (3). Here, we show that intestinal inflammation correlates with loss of LHPP, in DSS-treated mice and in inflammatory bowel disease (IBD) patients. Increased histidine phosphorylation was observed in intestinal epithelial cells (IECs), as determined by pHis immunofluorescence staining of colon samples from a colitis mouse model. However, ablation of Lhpp did not cause increased pHis or promote intestinal inflammation in physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine.