Role of the Two Structural Domains from the Periplasmic Escherichia coli Histidine-binding Protein HisJ

Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.     

Salmonella typhimurium histidine periplasmic permease mutations that allow transport in the absence of histidine-binding proteins.

Periplasmic transport systems consist of a membrane-bound complex and a periplasmic substrate-binding protein and are postulated to function by translocating the substrate either through a nonspecific pore or through specific binding sites located in the membrane complex. We have isolated mutants carrying mutations in one of the membrane-bound components of the histidine permease of Salmonella typhimurium that allow transport in the absence of both histidine-binding proteins HisJ and LAO (lysine-, arginine-, ornithine-binding protein). All of the mutations are located in a limited region of the nucleotide-binding component of the histidine permease, HisP. The mutants transported substrate in the absence of binding proteins only when the membrane-bound complex was produced in large amounts. At low (chromosomal) levels, the mutant complex was unable to transport substrate in the absence of binding proteins but transported it efficiently in the presence of HisJ. The alterations responsible for the mutations were identified by DNA sequencing; they are closely related to a group of hisP mutations isolated as suppressors of HisJ interaction mutations (G. F.-L. Ames and E. N. Spudich, Proc. Natl. Acad. Sci. USA 73:1877-1881, 1976). The hisP suppressor mutations behaved similarly to these newly isolated mutations despite the entirely different selection procedure. The results are consistent with the HisP protein carrying or contributing to the existence of a substrate-binding site that can be mutated to function in the absence of a binding protein.     

Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A. , Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem. 271, 21243-21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.     

Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP

The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.     

Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein,HisJ

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP‐cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L‐histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L‐His and many related naturally occurring compounds. Our data confirm that L‐His is the preferred ligand, but that 1‐methyl‐L‐His and 3‐methyl‐L‐His can also bind, while the dipeptide carnosine binds weakly and D‐histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L‐Arg, homo‐L‐Arg, and post‐translationally modified methylated Arg‐analogs also bind with reasonable avidity, with the exception of symmetric dimethylated‐L‐Arg. In contrast, L‐Lys and L‐Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein‐based reagentless biosensor.     

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae

In a search for components involved in Mn²⁺ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn²⁺ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn²⁺ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co²⁺, Ni²⁺, Zn²⁺, and Cu²⁺. The Mn²⁺ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn²⁺ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn²⁺ efflux. We propose that Hip1p is involved in Mn²⁺ transport, carrying out a function related to Mn²⁺ export.     

A novel mutant of the lac transport system of Escherichia coli.

A new type of lactose permease mutant, called lacY^f, does not actively transport the usual substrates; but it does facilitate the entry of β-galactosides into Escherichia coli K-12. The kinetics of facilitated entry, as assayed by hydrolysis of o-nitrophenyl-β-d-galactopyranoside by intact cells are identical to those observed with wild-type permease. However, the mutant permease activity is not affected by SH reagents or the substrate analog β-d-galactosyl-1-thio-β-d-galactopyranoside which strongly inhibit wild-type activity. Furthermore, the kinetics of formation of permease in the mutant following addition of inducer and the kinetics subsequent to removal of inducer differ strikingly from those observed in wild-type strains. The results are consistent with a block in the maturation of permease in the mutant resulting in the accumulation of a large amount of permease precursor. Studies of the $\text{lacY}{}^{\mathrm{+}}\text{lacY}{}^{\mathrm{f}}$ heterogenotes provide evidence for a subunit structure for the lactose permease.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase

Background

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK _a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.

Methodology/Principal Findings

Using His-HDX-MS, the pK _a values and the half-lives (t _1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP⁺ (DHFR-folate-NADP⁺) were determined. The results showed that the two parameters (pK _a and t _1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK _a, t _1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK _a and t _1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP⁺. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.

Conclusions/Significance

Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Histidine Residues in the Na+-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function,Modulating pH Sensitivity,Transporter Kinetics,Na+ Cooperativity,Conformational Stability,and Subcellular Localization

Na⁺-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His¹⁰⁹, His²⁰³, His²⁰⁶, His²⁶⁹, and His⁴¹³, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na⁺ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His⁴¹³, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na⁺ and loss of Na⁺ cooperativity, which leads to a decreased V_max without altering the transport K_m; (ii) exofacial histidine residues His²⁰³, His²⁰⁶, and His⁴¹³ may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K_m; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20–25 μmol/ml cell water per min. The apparent K_m is 3 · 10^-5. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one.In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyl-drazone (CCCP) or NaN₃ inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs.The specificity of the permease is very strict, only amino acids carrying an α-N-carbamyl group are strongly competitive inhibitors.The high concentration capacity of the cells and the lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Modification of Gene Expression and Virulence Traits in Streptococcus mutans in Response to Carbohydrate Availability

The genetic and phenotypic responses of Streptococcus mutans, an organism that is strongly associated with the development of dental caries, to changes in carbohydrate availability were investigated. S. mutans UA159 or a derivative of UA159 lacking ManL, which is the EIIAB component (EIIAB^Man) of a glucose/mannose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and a dominant effector of catabolite repression, was grown in continuous culture to steady state under conditions of excess (100 mM) or limiting (10 mM) glucose. Microarrays using RNA from S. mutans UA159 revealed that 174 genes were differentially expressed in response to changes in carbohydrate availability (P < 0.001). Glucose-limited cells possessed higher PTS activity, could acidify the environment more rapidly and to a greater extent, and produced more ManL protein than cultures grown with excess glucose. Loss of ManL adversely affected carbohydrate transport and acid tolerance. Comparison of the histidine protein (HPr) in S. mutans UA159 and the manL deletion strain indicated that the differences in the behaviors of the strains were not due to major differences in HPr pools or HPr phosphorylation status. Therefore, carbohydrate availability alone can dramatically influence the expression of physiologic and biochemical pathways that contribute directly to the virulence of S. mutans, and ManL has a profound influence on this behavior.     

Experimental and Theoretical Bases of Specific Affinity, a Cytoarchitecture-Based Formulation of Nutrient Collection Proposed To Supercede the Michaelis-Menten Paradigm of Microbial Kinetics

A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells⁻¹ h⁻¹ found were up to 10³ times smaller than those from a “Marinobacter arcticus ” isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on K_m was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with K_m as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.     

Spontaneous and reversible high-frequency frameshifts originating a phase transition in the carotenoid biosynthesis pathway of the phototrophic bacterium Rhodobacter sphaeroides 2.4.1

Summary The nucleotide sequence of the melB gene coding for the Na⁺(Li⁺)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with accession number X62101     

Nitrogen-regulated Ubiquitination of the Gap1 Permease of Saccharomyces cerevisiae

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH₄⁺ induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in the npi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH₄⁺-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH₄⁺ to mutants defective in endocytosis.     

Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 μg or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif⁺ plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his⁺ strain, and was also Nif^- on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.