Histidine-containing Phosphocarrier Protein Research Articles

Molecular Dynamics Simulations of HPr Proteins from a Thermophilic and a Mesophilic Organism: A Comparative Thermal Study.

The histidine-containing phosphocarrier (HPr) is a monomeric protein conserved in Gram-positive bacteria, which may be of mesophilic or thermophilic nature. In particular, the HPr protein from the thermophilic organism B. stearothermophilus is a good model system for thermostability studies, since experimental data, such as crystal structure and thermal stability curves, are available. However, its unfolding mechanism at higher temperatures is yet unclear at a molecular level. Therefore, in this work, we researched the thermal stability of this protein using molecular dynamics simulations, subjecting it to five different temperatures during a time span of 1 μs. The analyses of the structural parameters and molecular interactions were compared with those of the mesophilic homologue HPr protein from B. subtilis. Each simulation was run in triplicate using identical conditions for both proteins. The results showed that the two proteins lose stability as the temperature increases, but the mesophilic structure is more affected. We found that the salt bridge network formed by the triad of Glu3-Lys62-Glu36 residues and the salt bridge made up of Asp79-Lys83 ion pair are key factors to keep stable the thermophilic protein, maintaining the hydrophobic core protected and the structure packed. In addition, these molecular interactions neutralize the negative surface charge, acting as "natural molecular staples".

Identification of HPr kinase/phosphorylase inhibitors: novel antimicrobials against resistant Enterococcus faecalis

Enterococcus faecalis, a gram-positive bacterium, is among the most common nosocomial pathogens due to its limited susceptibility to antibiotics and its reservoir of the genes coding for virulence factors. Bacterial enzymes such as kinases and phosphorylases play important roles in diverse functions of a bacterial cell and, thus, are potential antibacterial drug targets. In Gram-positive bacteria, HPr Kinase/Phosphorylase (HPrK/P), a bifunctional enzyme is involved in the regulation of carbon catabolite repression by phosphorylating/dephosphorylating the histidine-containing phosphocarrier protein (HPr) at Ser46 residue. Deficiencies in HPrK/P function leads to severe defects in bacterial growth. This study aimed at identifying novel inhibitors of E. faecalis HPrK/P from a commercial compound library using structure-based virtual screening. The hit molecules were purchased and their effect on enzyme activity and growth of resistant E. faecalis was evaluated in vitro. Furthermore, docking and molecular dynamics simulations were performed to study the interactions of the hit compounds with HPrK/P. Among the identified hit molecules, two compounds inhibited the phosphorylation of HPr as well as significantly reduced the growth of resistant E. faecalis in vitro. These identified potential HPrK/P inhibitors open new research avenues towards the development of novel antimicrobials against resistant Gram-positive bacteria.

Impact of the Histidine-Containing Phosphocarrier Protein HPr on Carbon Metabolism and Virulence in Staphylococcus aureus.

Carbon catabolite repression (CCR) is a common mechanism pathogenic bacteria use to link central metabolism with virulence factor synthesis. In gram-positive bacteria, catabolite control protein A (CcpA) and the histidine-containing phosphocarrier protein HPr (encoded by ptsH) are the predominant mediators of CCR. In addition to modulating CcpA activity, HPr is essential for glucose import via the phosphotransferase system. While the regulatory functions of CcpA in Staphylococcus aureus are largely known, little is known about the function of HPr in CCR and infectivity. To address this knowledge gap, ptsH mutants were created in S. aureus that either lack the open reading frame or harbor a ptsH variant carrying a thymidine to guanosine mutation at position 136, and the effects of these mutations on growth and metabolism were assessed. Inactivation of ptsH altered bacterial physiology and decreased the ability of S. aureus to form a biofilm and cause infections in mice. These data demonstrate that HPr affects central metabolism and virulence in S. aureus independent of its influence on CcpA regulation.

Facilitated Protein Association via Engineered Target Search Pathways Visualized by Paramagnetic NMR Spectroscopy

Proteins assemble to form functional complexes via the progressive evolution of nonspecific complexes formed by transient encounters. This target search process generally involves multiple routes that lead the initial encounters to the final complex. In this study, we have employed NMR paramagnetic relaxation enhancement to visualize the encounter complexes between histidine-containing phosphocarrier protein and the N-terminal domain of enzyme I anddemonstrate that protein association can be significantly enhanced by engineering on-pathways. Specifically, mutations in surface charges away fromthe binding interface can elicit new on-pathway encounter complexes, increasing their binding affinity by an order of magnitude. The structure of these encounter complexes indicates that such on-pathways extend the built-in target search process of the native protein complex. Furthermore, blocking on-pathways by countering mutations reverts their binding affinity. Our study thus illustrates that protein interactions can be engineered by rewiring the target search process.

Mu insertion in feuD triggers the increase in nisin immunity in Lactococcus lactis subsp. lactis N8.

This study aims to explore how feuD mutation triggered the increase in nisin immunity of Lactococcus lactis L58, which was proven to be a feuD::Em-Mu mutant of Lc. lactis N8. The significant difference genes of Lc. lactis L58 and Lc. lactis N8 were compared at transcription and protein levels. Analysis revealed that the feuD mutation induced decrease in histidine-containing phosphocarrier protein PtsH (HPr) and increase in thioredoxin reductase TrxB (TR). Determination of iron concentration and cytoplasmic membrane potential (MP) showed the iron concentration decreased around 10% and the MP decreased approx. 14% in Lc. lactis L58. The increase in nisin immunity was dominated by TR up-expression by two main mechanisms in Lc. lactis L58. First, the TR-TRX (thioredoxin reductase) system changed the composition of cytoplasmic membrane by regulating the lipid metabolism to enhance the cells' resistance to nisin. Second, iron starvation stress induced decrease in MP; hence, the binding affinity of nisin to lipid II of Lc. lactis L58 decreased, which, in turn, increased the nisin immunity. The knowledge on regulation mechanism of nisin immunity was enriched, and the theoretical basis for improving nisin production in engineering strain could be provided.

Structural basis for the sequestration of the anti-σ(70) factor Rsd from σ(70) by the histidine-containing phosphocarrier protein HPr.

Histidine-containing phosphocarrier protein (HPr) is a general component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) involved in the phosphorylation-coupled transport of numerous sugars called PTS sugars. HPr mainly exists in a dephosphorylated form in the presence of PTS sugars in the medium, while its phosphorylation increases in the absence of PTS sugars. A recent study revealed that the dephosphorylated form of HPr binds and antagonizes the function of the antisigma factor Rsd. This anti-sigma factor sequesters the housekeeping sigma factor σ(70) to facilitate switching of the sigma subunit on RNA polymerase from σ(70) to the stress-responsive sigma factor σ(S) in stationary-phase cells. In this study, the structure of the complex of Rsd and HPr was determined at 2.1 Å resolution and revealed that the binding site for HPr on the surface of Rsd partly overlaps with that for σ(70). The localization of the phosphorylation site on HPr at the binding interface for Rsd explains why phosphorylation of HPr abolishes its binding to Rsd. The mutation of crucial residues involved in the HPr-Rsd interaction significantly influenced the competition between HPr and σ(70) for binding to Rsd both in vitro and in vivo. The results provide a structural basis for the linkage of global gene regulation to nutrient availability in the external environment.

The Phosphoenolpyruvate:Sugar Phosphotransferase System Is Involved in Sensitivity to the Glucosylated Bacteriocin Sublancin

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin.

HPr antagonizes the anti-σ 70 activity of Rsd in Escherichia coli

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

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.

Crystal structure of histidine-containing phosphocarrier protein from Thermoanaerobacter tengcongensis MB4 and the implications for thermostability

Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms, and therefore enables these organisms to survive at extreme temperatures. Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms, the structural basis of protein thermostability is not yet fully understood. The histidine-containing phosphocarrier (HPr) protein, a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species, is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors. In this study, the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified. Crystal structure with good quality has been determined at 2.3 Å resolution, which provides a firm foundation for exploring the thermostable mechanism. However, it shows that the crystal structure is conserved and no clue can be obtained from this single structure. Furthermore, detailed comparison of sequence and structure with the homologs from meso- or thermophilic bacteria shows no obvious rule for thermostability, but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr. Thus, mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria. Using site-directed mutations and the circular dichroism method, thermostability is evaluated, and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses, indicating that mutations cause instability in the HPrs. Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability, and is critical in engineering efficient enzymes that can work at a high temperature.

Evidence that small proteins translocate through silicon nitride pores in a foldedconformation

The interaction of three proteins (histidine-containing phosphocarrier protein, HPr,calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiNx) membranes has been studied. The proteins which have a net negative charge wereelectrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm.The % blockade current and event duration were measured at three different voltages. For atranslocation event it was expected that the % block would be constant with voltage whilstthe event duration would decrease with increasing voltage. On the basis of these criteria,we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas upto 40% of CaM molecules can translocate the 7 nm pore as can a majority ofHPr molecules, with some translocations being observed for the 5 nm pore. Fortranslocation events the magnitude of the % blockade current is consistent with afolded conformation of the proteins surrounded by a hydration shell of 0.5–1.0 nm.

Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements.

Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Gamma(2)) rates measured on (15)N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.

Increasing protein stability by improving beta‐turns

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability.

Single-molecule protein unfolding in solid state nanopores.

We use single silicon nitride nanopores to study folded, partially folded, and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of beta-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins. Our results show that the nanopore translocation signals are sensitive enough to distinguish the folding state of a protein and distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain that transiently stalls in the nanopore due to the primary sequence of charges.

Folded and Unfolded Single Proteins Analyzed by Their Solid State Nanopore Translocation Dynamics

The translocation of biological polymers through individual nanometer-scale pores is vital to cellular function and has great potential for technological applications in protein or nucleic acid measurements and identification. Research into this area has been focussed on characterizing the physics of translocation through voltage-biased nanopores and exploiting it to identify or sequence biological polymers. Here we show that the DNA-calibrated translocation signals of s-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids present in the pore when it stalls due to its primary charge. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation suggesting that physiologically relevant potentials can unfold proteins. Our results suggest that the nanopore translocation physics and signals are sensitive enough to distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain and the primary sequence of charges.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Effects of Histidine Protonation and Phosphorylation on Histidine-Containing Phosphocarrier Protein Structure, Dynamics, and Physicochemical Properties

Previous structural studies of the histidine-containing phosphocarrier protein (HPr) have shown that active site residue His15 can adopt two distinct conformations which were termed OPEN and CLOSED. Using molecular dynamics simulations and protonation probability calculations, we were able to show that these two conformations correspond to different protonation forms of the histidine ring. The CLOSED-to-OPEN transition requires His15 to adopt a conformation with higher energy, which is compensated by the favorable energetic consequences of protonation. Calculations of the conformational energy of His15 show that HPr exists mainly in the CLOSED form at pH 7. The very low apparent pKa value (3.2-4.5) of the CLOSED conformation and the fact that the imidazole ring of residue 15 is primarily unprotonated at Ndelta1 at neutral pH ensure that His15 is ideally primed to be specifically phosphorylated at Ndelta1. In contrast to unphosphorylated HPr, the phosphorylated form exhibits no conformational transitions, and the CLOSED state is stable even for the protonated imidazole ring due to favorable interactions between the phosphate group and the backbone of Ala16 and Arg17. These observations from MD simulations are confirmed by a simple four-microstate model which can explain both the pH-dependent conformational change of unphosphorylated HPr and the conformational rigidity of phosphorylated HPr. Our study suggests that the predominant CLOSED conformation is relevant for HPr function in the phosphotransfer reaction, while the OPEN form of unphosphorylated HPr might be important for its additional regulatory function, in which an OPEN conformation of His15 is recognized by the transcriptional regulator CcpA.

Dynamics of Tryptophan in the Histidine-Containing Phosphocarrier Protein of Streptomyces coelicolor: Evidence of Multistate Equilibrium Unfolding

The nanosecond dynamics of the single tryptophan, Trp10, of HPr from Streptomyces coelicolor, HPrsc, has been monitored at different pHs. Time-resolved fluorescence methods and DOSY measurements have been used to map the compactness of the protein. At low pHs, where a molten globule-like species has been described, the correlation times from fluorescence showed an abrupt change as the pH was increased. When the protein was folded (above pH 4), two correlation times were observed, which remained practically constant up to pH 9.5. The long correlation time, around 7.5 ns, corresponds to the global rotational motion of the protein, since this value is in agreement with that determined theoretically from hydrodynamic measurements. The short correlation time, around 1.4 ns, must report on fast movements of the protein segment containing the tryptophan residue. On the other hand, fluorescence lifetimes showed the same abrupt change as the correlation times at low pH, but, in addition, a sigmoidal change with a pKa approximately 4.3 was also observed. On the basis of the modeled structure of HPrsc, this last transition could be due to the proximity of Glu12 to Trp10. The changes monitored by the fluorescence lifetimes agree with those observed previously by steady-state fluorescence, CD, and ANS binding experiments. Taken together, these data suggest a multistate equilibrium during folding of HPrsc starting from low pHs.

Crystal structure of a transcription regulator (TM1602) from Thermotoga maritima at 2.3 Å resolution

Crystal structure of a transcription regulator (TM1602) from <i>Thermotoga maritima</i> at 2.3 Å resolution

AUREMOL-RFAC-3D, combination of R-factors and their use for automated quality assessment of protein solution structures

We present here the computer program AUREMOL-RFAC-3D that is a generalization of the previously published program RFAC for the fully automated estimation of residual indices (R-factors) from 2D NOESY spectra. It is part of the larger AUREMOL software package (www.auremol.de). RFAC-3D calculates R-factors directly from two-dimensional homonuclear NOESY spectra as well as from three-dimensional (15)N or (13)C edited NOESY-HSQC spectra and thus extends the application range to larger proteins. The fully automated method includes automated peak picking and integration, a Bayesian noise and artifact recognition and the use of the complete relaxation matrix formalism. To enhance the reliability of the calculated R-factors the method is also generalized to calculate combined R-factors from a set of 2D and 3D-spectra. For an optimal combination of the information derived from different sources a plausible formalism had to be derived. In addition, we present a novel direct R-factors based measure that correlates an R-factors as defined in this paper to the root mean square deviation of the actual structure from the optimal structure. The new program has been successfully tested on the histidine containing phosphocarrier protein (HPr) from Staphylococcus carnosus and on the Ras-binding domain (RBD) of the Ral guanine-nucleotide dissociation stimulation factor (RalGDS).

A Thermodynamic Comparison of HPr Proteins from Extremophilic Organisms

A thermodynamic stability study of five histidine-containing phosphocarrier protein (HPr) homologues derived from organisms inhabiting diverse environments is described. These HPr homologues are from Bacillus subtilis (Bs), Streptococcus thermophilus (St), Bacillus staerothermophilus (Bst), Bacillus halodurans (Bh), and Oceanobacillus iheyensis (Oi). Analyses of solvent and thermal denaturation experiments provide the cardinal thermodynamic parameters, like deltaG, deltaH, deltaS, T(m), and deltaC(p), that characterize the conformational stability for each homologue. The homologue from Bacillus staerothermophilus (BstHPr) was established as the most thermostable homologue and also the homologue with highest deltaG at all temperatures. A good correlation between habitat temperature of the organism and thermal stability of the protein is also seen. Stability curves (deltaG vs T) for every homologue are also reported; these reveal very similar deltaC(p) and temperature of maximum stability (T(S)) values for all HPr homologues. Stability curves show that the higher thermal stability of some homologues is not a result of change in curvature of the curve or a shift to higher temperature, but rather a displacement of the stability curves to higher deltaG values. Stability curves also allowed estimation of deltaG at habitat temperature of the organisms, and we find good agreement between homologues. Electrostatic contributions to stability of each homologue were investigated by measuring stability as a function of varying pH and NaCl concentration, and our results suggest that most HPr homologues share similar electrostatic contributions to stability.