Histidine-containing Protein Research Articles

Metabolomic profiling of bacterial biofilm: trends, challenges, and an emerging antibiofilm target.

Biofilm-related infections substantially contribute to bacterial illnesses, with estimates indicating that at least 80% of such diseases are linked to biofilms. Biofilms exhibit unique metabolic patterns that set them apart from their planktonic counterparts, resulting in significant metabolic reprogramming during biofilm formation. Differential glycolytic enzymes suggest that central metabolic processes are markedly different in biofilms and planktonic cells. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is highly expressed in Staphylococcus aureus biofilm progenitors, indicating that changes in glycolysis activity play a role in biofilm development. Notably, an important consideration is a correlation between elevated cyclic di-guanylate monophosphate (c-di-GMP) activity and biofilm formation in various bacteria. C-di-GMP plays a critical role in maintaining the persistence of Pseudomonas aeruginosa biofilms by regulating alginate production, a significant biofilm matrix component. Furthermore, it has been demonstrated that S. aureus biofilm development is initiated by several tricarboxylic acid (TCA) intermediates in a FnbA-dependent manner. Finally, Glucose 6-phosphatase (G6P) boosts the phosphorylation of histidine-containing protein (HPr) by increasing the activity of HPr kinase, enhancing its interaction with CcpA, and resulting in biofilm development through polysaccharide intercellular adhesion (PIA) accumulation and icaADBC transcription. Therefore, studying the metabolic changes associated with biofilm development is crucial for understanding the complex mechanisms involved in biofilm formation and identifying potential targets for intervention. Accordingly, this review aims to provide a comprehensive overview of recent advances in metabolomic profiling of biofilms, including emerging trends, prevailing challenges, and the identification of potential targets for anti-biofilm strategies.

Surfactin, a quorum sensing signal molecule, globally affects the carbon metabolism in Bacillus amyloliquefaciens

Surfactin, a quorum sensing signal molecule, is correlated with carbon metabolism in Bacillus amyloliquefaciens. In the present work, we found that mutation of srfA (ΔsrfA) led to an obviously changed carbon metabolism in B. amyloliquefaciens. Firstly, the PTS-glucose system was significantly increased as a feedback to glucose exhaustion. Secondly, the basic carbon metabolism such as glycolysis and TCA cycle was obviously weakened in ΔsrfA. Thirdly, the global regulator of CcpA (carbon catabolite protein A) and P ~ Ser46-HPr (seryl-phosphorylated form of histidine-containing protein) to mediate the CcpA-dependent CCR (carbon catabolite repression) were not increased, but the ability to use extracellular non- and less-preferred carbon sources was down-regulated in ΔsrfA. Fourthly, the carbon overflow metabolism such as biosynthesis of acetate was enhanced while biosynthesis of acetoin/2,3-butanediol and branched-chain amino acids were weakened in ΔsrfA. Finally, ΔsrfA could use most of non- and less-preferred carbon sources except for fatty acids, branched chain amino acids, and some organic acids (e.g. pyruvate, citrate and glutamate) after glucose exhaustion. Collectively, surfactin showed a global influence on carbon metabolism in B. amyloliquefaciens. Our studies highlighted a way to correlate quorum sensing with carbon metabolism via surfactin in Bacillus species.

Systematic analysis of an evolved Thermobifida fusca muC producing malic acid on organic and inorganic nitrogen sources.

Thermobifida fusca is a thermophilic actinobacterium. T. fusca muC obtained by adaptive evolution preferred yeast extract to ammonium sulfate for accumulating malic acid and ammonium sulfate for cell growth. We did transcriptome analysis of T. fusca muC on Avicel and cellobiose with addition of ammonium sulfate or yeast extract, respectively by RNAseq. The transcriptional results indicate that ammonium sulfate induced the transcriptions of the genes related to carbohydrate metabolisms significantly more than yeast extract. Importantly, Tfu_2487, encoding histidine-containing protein (HPr), didn’t transcribe on yeast extract at all, while it transcribed highly on ammonium sulfate. In order to understand the impact of HPr on malate production and cell growth of the muC strain, we deleted Tfu_2487 to get a mutant strain: muCΔ2487, which had 1.33 mole/mole-glucose equivalent malate yield, much higher than that on yeast extract. We then developed an E. coli-T. fusca shuttle plasmid for over-expressing HPr in muCΔ2487, a strain without HPr background, forming the muCΔ2487S strain. The muCΔ2487S strain had a much lower malate yield but faster cell growth than the muC strain. The results of both mutant strains confirmed that HPr was the key regulatory protein for T. fusca’s metabolisms on nitrogen sources.

Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.

The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG). However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr) could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P) with no need to add fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc-6-P) in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA) and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (K D) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex.To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

On-particle detection of Plasmodium falciparum histidine-rich protein II by a “switch-on” iridium(III) probe

The need for robust reagents for biomarker detection has become an increasing necessity in designing point-of-care diagnostics. We report a non-emissive, cyclometalated iridium(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which, on coordination to a histidine-containing protein bound to the surface of a magnetic particle, elicits a rapid, long-lived phosphorescent signal. The interactions between Ir1 and numerous other amino acids were examined for activity, but only the addition of histidine resulted in a four orders of magnitude enhancement in signal intensity. Buffer conditions (pH and temperature) and composition (coordinating vs. non-coordinating and ionic strength) were optimized to achieve maximum signal and stability of Ir1. The activity of the probe under optimized conditions was validated with BNT-II, a histidine-containing branched peptide mimic of the malarial biomarker Plasmodium falciparum histidine-rich protein II (PfHRPII). By comparing Ir1 binding to BNT-II versus l-histidine, steric and quenching effects were noted in the peptide. Despite these deviations from ideal conditions, signal response reached saturation with both BNT-II and recombinant HRPII (rcHRPII). When immobilized on the surface of a 50μM magnetic agarose particles, the limit of detection of rcHRPII was 14.5nM. The robust signal response of this inorganic probe lends itself to future applications in on-particle enzyme-linked immunosorbent assay (ELISA)-based assays.

Carbon catabolite repression in Thermoanaerobacterium saccharolyticum

BackgroundThe thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.ResultsWe report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.ConclusionCo-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis

BackgroundIn Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.ResultsThe transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed.ConclusionsCertain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified.

Solution Structure of the IIAChitobiose-HPr Complex of the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System

The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIA(Chb)-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.

CcpA forms complexes with CodY and RpoA in Bacillus subtilis

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the α-subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose, whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N-terminal domain (αNTD) and C-terminal domain (αCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by surface plasmon resonance. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are in the range 5-8 μm. CcpA and CodY form a loose complex with a K(D) of 60 μm. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Crystal Structure of a Putative Histidine-containing Phosphotransfer Protein from Oryza sativa

Os09g0567400 codes for a hypothetical protein from Oryza sativa that is annotated as the “Histidine-containing phosphotransfer (Hpt) protein”. Hpt domain is a protein module with a histidine residue mediating phosphotransfer reaction in the histidine-aspartate phosphorelay system. We report here the crystal structure and analysis of Os09g0567400.

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

The histidine-containing protein (HPr) is the energy coupling protein of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes the transport of carbohydrates in bacteria. In Bacillus subtilis and close relatives, global regulation of carbon catabolite control occurs on the binding of the complex of CcpA (catabolite control protein A) and P-Ser-HPr (seryl-phosphorylated form of HPr) to the catabolite responsive elements (cre) of the target operons, the constituent genes of which are roughly estimated to number 300. The complex of CcpA and P-Ser-HPr triggers the expression of several genes involved in the formation of acetate and acetoin, major extracellular products of B. subtilis grown on glucose. It also triggers the expression of an anabolic operon (ilv-leu) involved in the biosynthesis of branched-chain amino acids, which subsequently leads to cell propagation. On the other hand, this complex represses many genes and operons, which include an entrance gene for the TCA cycle (citZ), several transporter genes for TCA cycle-intermediates, some respiration genes, and many catabolic and anabolic genes involved in carbon, nitrogen, and phosphate metabolism, as well as for certain extracellular enzymes and secondary metabolites. Furthermore, these bacteria have CcpA-independent catabolite regulation systems, each of which involves a transcriptional repressor of CggR or CcpN. CggR and CcpN are derepressed under glycolytic and gluconeogenic growth conditions, and enhance glycolysis and gluconeogenesis respectively. Another CcpA-independent catabolite repression system involves P-His-HPr (histidyl-phosphorylated form of HPr). P-His-HPr phosphorylates and activates glycerol kinase, whose product is necessary for antitermination of the glycerol utilization operon through GlpP, the antiterminators (LicT and SacT, Y) of several operons for the utilization of less-preferred PTS-sugars, and some transcriptional activators such as LevR for the levan utilization operon. This phosphorylation is reduced due to the decreased level of P-His-HPr during active transport of a preferred PTS-carbohydrate such as glucose, resulting in catabolite repression of the target operons.Thus CcpA-dependent and independent networks for carbon metabolism play a major role in the coordinate regulation of catabolism and anabolism to ensure optimum cell propagation in the presence and the absence of a preferred PTS-carbohydrate.

Structural investigation of a phosphorylation-catalyzed, isoaspartate-free, protein succinimide: crystallographic structure of post-succinimide His15Asp histidine-containing protein.

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through the high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.

Visualization of Transient Ultra-Weak Protein Self-Association in Solution Using Paramagnetic Relaxation Enhancement

Ultra-weak macromolecular self-association is exceptionally difficult to both detect and visualize using conventional biophysical techniques owing to the very low population of the associated species, yet such weak intermolecular interactions coupled with nucleation events play an important role in driving spontaneous self-assembly to form higher-order architectures (such as crystals, viral capsids, and amyloid fibrils). In this article, we detect and characterize transient, ultra-weak self-association (KD >or= 15 mM) involving the histidine-containing protein HPr by means of paramagnetic relaxation enhancement (PRE), using EDTA-Mn2+ conjugated at three separate sites (E5C, E25C, and E32C, one at a time). Large intermolecular PRE effects, above the background observed with hydroxylamine-EDTA-Mn2+ as a control, are observed for two of the three paramagnetically labeled sites, E5C and E32C. The extent of self-association can be modulated (significantly reduced) by increasing the ionic strength or by the introduction of a negative charge (S46D mutation) within a positively charged surface patch, and abolished upon the addition of the N-terminal domain of enzyme I (EIN) to form a specific EIN-HPr complex. The PRE profiles observed for E5C and E32C can be fitted simultaneously and accounted for quantitatively using conjoined rigid body/torsion angle dynamics-simulated annealing with an ensemble of states to represent the distribution of one molecule of HPr relative to its partner.

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.

The transcription regulator RbsR represents a novel interaction partner of the phosphoprotein HPr‐Ser46‐P in Bacillus subtilis

Histidine-containing protein (HPr) is a central metabolic sensor in low-GC Gram-positive bacteria and plays a dual role in sugar uptake by the phosphoenolpyruvate-sugar phosphotransferase system and in transcriptional control during carbon catabolite repression. The latter process is mediated by interaction between HPr and the carbon catabolite repression master transcription regulator, carbon catabolite protein A (CcpA), a member of the LacI-GalR family of DNA-binding proteins. We investigated, with a combination of computational and experimental tools, whether HPr can also interact with other transcriptional regulators. To allow rapid identification of paralogous LacI-GalR family members that might interact with HPr in a similar fashion to CcpA, a structure-based computational approach was developed which relies on the analysis of the similarity of protein-protein interfaces between different complexes. A key element of this method is an empirical pair potential derived from a group of orthologous complexes and subsequently used to identify paralogs that exhibit similar properties of their protein interfaces. Application of this method to the family of LacI-GalR repressors in Bacillus subtilis predicted the ribose operon repressor (RbsR) as a novel interaction partner of HPr. This interaction was subsequently confirmed experimentally and suggests that HPr plays an even larger role in transcriptional control than previously expected.

Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation

In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser46-P or Crh-Ser46-P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser46-P more weakly than HPr-Ser46-P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser46-P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser46-P)-DNA complex. The structure reveals that Crh-Ser46-P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser46-P. The reduced affinity of Crh-Ser46-P for CcpA as compared with that of HPr-Ser46 P is explained by weaker Crh-Ser46-P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix alpha 2 of the second contact region (contact region II) of Crh-Ser46-P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA.

Molecular dynamics simulations of HPr under hydrostatic pressure.

The histidine-containing protein (HPr) plays an important role in the phosphotransferase system (PTS). The deformations induced on the protein structure at high hydrostatic pressure values (4, 50, 100, 150, and 200 MPa) were previously (H. Kalbitzer, A. Görler, H. Li, P. Dubovskii, A. Hengstenberg, C. Kowolik, H. Yamada, and K. Akasaka, Protein Science 2000, Vol. 9, pp. 693-703) analyzed by NMR experiments: the nonlinear variations of the amide chemical shifts at high pressure values were supposed to arise from induced shifts in the protein conformational equilibrium. Molecular dynamics (MD) simulations are here performed, to analyze the protein internal mobility at 0.1 MPa, and to relate the nonlinear variations of chemical shifts observed at high pressure, to variations in conformational equilibrium. The global features of the protein structure are only slightly modified along the pressure. Nevertheless, the values of the Voronoi residues volumes show that the residues of alpha-helices are more compressed that those belonging to the beta-sheet. The alpha-helices are also displaying the largest internal mobility and deformation in the simulations. The nonlinearity of the 1H chemical shifts, computed from the MD simulation snapshots, is in qualitative agreement with the nonlinearity of the experimentally observed chemical shifts.