A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mutation hisJ5625 has altered the histidine-binding protein J of Salmonella typhimurium such that histidine transport is impaired, even though binding of histidine by the J protein is unimpaired [Kustu, S.G., & Ames, G.F. (1974) J. Biol. Chem. 249, 6976--6983]. We have determined by protein analytical methods that the only effect of this mutation has been the substitution of a cysteine residue for an arginine at a site in the interior of the polypeptide chain. This arginine residue is therefore potentially essential for the transport function of the protein. The mutant protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis more slowly than the wild type protein, as if its molecular weight were greater by as much as 2000. Since this behavior is apparently due to a single amino acid replacement, a molecular weight difference even between two closely related proteins should not be inferred solely on the basis of sodium dodecyl sulfate gel electrophoresis.     

Evidence for a common mechanism for the insertion of the Tn10 transposon and for the generation of Tn10-stimulated deletions

Summary Mutations in and near the Salmonella typhimurium histidine transport operon were generated by insertion of the translocatable tetracycline-resistance element Tn10. Deletion mutants affecting histidine transport genes were subsequently isolated in several of the Tn10-containing strains. Tn10 insertions in hisJ occurred preferentially at one site, designated site A. This same site was also the preferential endpoint of deletions originating from Tn10 insertions at two neighboring sites. Thus, Tn10 insertion and Tn10-stimulated deletion formation appear to involve a common DNA-recogition step.     

A high-resolution 1H-NMR investigation of the histidine-binding protein J of Salmonella Typhimurium: Substrate-induced conformational changes

High-resolution ¹ H-NMR spectroscopy at 600 MHz has been used to investigate the conformational transitions of the histidine-binding protein J of Salmonella Typhinmrium in solution as a function of pH and of l-histidine concentration. The dissociation constant for the binding of l-histidine to histidine-binding protein J increases from 6.0 × 10^?8 to 5.1 × 10^?7 M in going from pH 5.57 to 8.00. The conformation of this protein as observed by ¹H-NMR also changes over this range of pH. However, when l-histidine is bound, the changes in conformation with pH are much smaller. Also, the pk for the single histidyl residue in histidine-binding protein J changes from 6.75 in the absence of l-histidine to 6.52 when l-histidine is bound. Earlier work in this laboratory resulted in the identification of several proton resonances believed to be at or near the l-histidine-binding site. Two of these resonances have been assigned to a tyrosine and the single histidyl residue in the histidine-binding protein J molecule.     

A proton nuclear magnetic resonance investigation of histine-binding protein J of salmonella typhimurium: A model for transport of L-histidine across cytoplasmic membrane

Genetic evidence suggests that the high-affinity L-histidine transport in Salmonella typhimurium requires the participation of a periplasmic binding protein (histidine-binding protein J) and two other proteins (P and Q proteins). The histidine-binding protein J binds L-histidine as the first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution proton nuclear magnetic resonance spectroscopy at 600 MHz is used to investigate the conformations of this protein in the absence and presence of substrate. Previous nuclear magnetic resonance results reported by this laboratory have shown that there are extensive spectral changes in this protein upon the addition of L-histidine. When resonances from individual amino acid residues of a protein can be resolved in the proton nuclear magnetic resonance spectrum, a great deal of detailed information about substrate-induced structural changes can be obtained. In order to gain a deeper insight into the nature of these structural changes, deuterated phenylalanine or tyrosine has been incorporated into the bacteria. Proton nuclear magnetic resonance spectra of selectively deuterated histidine-binding protein J were obtained and compared to the normal protein. Several of the proton resonances have been assigned to the various aromatic amino acid residues of this protein. A model for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium is proposed. This model, which is a version of the pore model, assumes that both P and Q proteins are membrane-bound and that the interface between these two proteins forms the channel for the passage of substrate. The histidine-binding protein J serves as the “key” for the opening of the channel for the passage of L-histidine. In the absence of substrate, this channel or gate is closed owing to a lack of appropriate interactions among these three proteins. The channel can be opened upon receiving a specific signal from the “key”; namely, the substrate-induced conformational changes in the histidine-binding protein J molecule. This model is consistent with available experimental evidence for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium.     

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.     

A mutational hot-spot in the hisM gene of the histidine transport operon in Salmonella typhimurium is due to deletion of repeated sequences and results in an altered specificity of transport

Summary Duplicated sequences within hisM, a gene coding for a membrane-bound component of histidine transport, result in frequent deletions which, being in frame, allow production of an altered protein with aparent changed specificity of transport. While the wild-type transport system does not transport L-histidinol but does transport L-histidine and several of its analogs, the hisM deletion mutants do not transport the latter compounds but do transport L-histidinol. These results are interpreted as supporting the hypothesis (Ames and Higgins 1983) that transport through periplasmic systems involves binding of the substrate by the cytoplasmic membrane-bound components.     

Overproduction of the membrane-bound components of the histidine permease from Salmonella typhimurium: identification of the M protein

The periplasmic histidine permease of Salmonella typhimurium is composed of a soluble histidine-binding protein and three membrane-bound components. These latter are produced in very small amounts and only two, the Q and the P protein, have been previously identified. This paper describes the construction of a plasmid carrying the hisQ, hisM, and hisP genes under the control of the lambda PL promoter, thus allowing great overproduction of those gene products. The M protein has been identified in such overproducing strains and its nature confirmed by constructing in vitro hisM deletions within the plasmid. With these results the identification of all components of the histidine permease has been completed.     

Determination of a region of the HisJ binding protein involved in the recognition of the membrane complex of the histidine transport system of Salmonella typhimurium

Site-directed mutagenesis has been utilized to examine the nature of the interaction of the histidine-binding protein (HisJ) with the membrane-bound components of the histidine transport system. In order to examine a region of the HisJ protein involved in the interaction with the membrane components, a number of charged amino acids in the vicinity of the genetically isolated interaction mutant hisJ5625 (R176C) were mutated. It was found that residues Asp171, Arg176, and Asp178 could be independently altered without affecting the histidine-binding affinity of the HisJ protein. However, the alteration of residues Asp171 and Arg176 greatly reduced the interaction of the HisJ protein with the membrane protein complex, whereas altering residue Asp178 had no effect on this interaction. Simultaneously, altering residues Asp183 and Glu184 resulted in a completely defective protein. The ability of a his-J5625 suppressor HisP protein (HisP(T205A)) to suppress the newly created site-directed mutants was also examined. This suppressor demonstrated specificity toward the amino acid present at position 176 and was also able the suppress the mutation created at position 171.     

Cloning of the histidine transport genes from salmonella typhimurium and characterization of an analogous transport system in escherichia coli

The genes for the well-characterized high-affinity histidine transport system of S typhimurium have been cloned in λgt4. Genetic and physiological analyses of the analogous transport system of E coli were undertaken in order that available λ vectors, recombinant DNA techniques, and a genetic selection for transport function might be used to isolate the Salmonella genes. The presence of the transport genes on a 12.4 Kb cloned DNA fragment has been confirmed (1) genetically, by complementation studies; (2) physiologically, by the rates of histidine uptake by bacteria containing this DNA; and (3) by demonstrating that the cloned DNA codes for the previously identified transport proteins J and P. The isolated fragment carries the entire transport operon, the argT gene and the ubiX locus, but neither the purF gene nor the ack/pta loci.     

Nuclear magnetic resonance and fluorescence studies of substrate-induced conformational changes of histidine-binding protein J of Salmonella typhimurium.

The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate. Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine. In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium. There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J. Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine. These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate. The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates.     

RNA Affinity for Molecular L-Histidine; Genetic Code Origins

Selection for affinity for free histidine yields a single RNA aptamer, which was isolated 54 times independently. This RNA is highly specific for the side chain and binds protonated L-histidine with 10²−10³-fold stereoselectivity and a dissociation constant (K_D) of 8–54 μM in different isolates. These histidine-binding RNAs have a common internal loop–hairpin loop structure, based on a conserved RAAGUGGGKKN_0–36 AUGUN_0–2AGKAACAG sequence. Notably, the repetitively isolated sequence contains two histidine anticodons, both implicated by conservation and chemical data in amino acid affinity. This site is probably the simplest structure that can meet our histidine affinity selection, which strengthens experimental support for a “stereochemical” origin of the genetic code.[Reviewing Editor: Niles Lehman]     

Interaction of thiourea with band 3 in human red cell membranes

Summary Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent,pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore,pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence ofpCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea,pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved.     

Functional domains of the gastric HK ATPase

The gastric H⁺ + K⁺ ATPase is a member of the phosphorylating class of transport ATPase. Based on sequence homologies and CHO content, there may be ab subunit associated with the catalytic subunit of the H⁺ + K⁺ ATPase. Its function, if present, is unknown. The pump catalyzes a stoichiometric exchange of H⁺ for K⁺, but is also able to transport Na⁺ in the forward direction. This suggests that the transport step involves hydronium rather than protons. The initial binding site is likely to contain a histidine residue to account for the high affinity of the cellular site. The extracellular site probably lacks this histidine, so that a low affinity for hydronium allows release into a solution of pH 0.8. Labelling with positively charge, luminally reactive reagents that block ATPase and pump activity has shown that a region containing H5 and H6 and the intervening luminal loop is involved in necessary conformational changes for normal pump activity. The calculated structure of this loop shows the presence of ana helical,b turn, andb strand sector, with negative charges close to the membrane domain. This sector provides a possible site of interaction of drugs with the H⁺ + K⁺ ATPase, and may be part of the K⁺ pathway in the enzyme.Emory University, Atlanta, Georgia.     

Functional characteristics of the cardiac sarcolemmal monocarboxylate transporter

Summary We have previously shown that a mechanism for transportingl-lactate is located in cardiac sarcolemmal membranes (Am. J. Physiol. 252:C483–C489, 1987). This mechanism has now been shown to transport pyruvate also. The transporter recognizes a wide range of monocarboxylic acids with chain lengths of three to six carbons, as evidenced by their ability to inhibitl-lactate uptake into sarcolemmal vesicles. The ability of the monocarboxylate analogues to inhibit depends strongly on the nature of substituents, particularly at the second carbon.l-lactate and pyruvate transport are not affected by dicarboxylates other than oxaloacetate. The transporter is inhibited by the protein modifiers diethylpyrocarbonate, dinitrofluorobenzene, and phenylisothiocyanate. Diethylpyrocarbonate inhibition is not reversed by hydroxylamine, nor is dinitrofluorobenzene inhibition reversed by thiol reagents, suggesting that the target residues are not histidine, or tyrosine or cysteine, respectively. Several monocarboxylates effectively protect the transporter from inhibition by the modifying reagents, suggesting that the modified residue(s) may be at or near the binding site. Alternatively, the target amino acid(s) in the transport protein may become inaccessible due to a conformation change triggered by the substrate analogues. Overall, the results suggest that a sensitive free amino group, associated with substrate binding, is attacked by the protein-modifying reagents.     

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297–4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.     

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.     

An analysis of the structure of the product of the rbsA gene of Escherichia coli K12

The predicted amino acid sequence of rbsA, a gene from the high affinity ribose transport operon (rbs) of Escherichia coli K12, is homologous to the products of hisP, malK, and pstB, components of the histidine, maltose, and phosphate high affinity transport operons. The recent finding by Hobson et al. (Hobson, A. C., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7333-7337) that the hisP and malK products bind ATP suggests that these four gene products may be involved in coupling the energy from ATP to drive the active transport in their respective transport systems. Each gene product contains a sequence of glycine and basic residues which are characteristic of an ATP-binding site (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951). Interestingly the N- and C-terminal halves of rbsA are also homologous, suggesting that a primordial gene duplication and subsequent fusion of the products occurred.     

Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine

ATP phosphoribosyltransferase (ATP-PRT) catalyzes the condensation of ATP and PRPP at the first step of histidine biosynthesis and is regulated by a feedback inhibition from product histidine. Here, we report the genetic and biochemical characterization of such an enzyme, HisG_Cg, from Corynebacterium glutamicum, including site-directed mutagenesis of the histidine-binding site for the first time. Gene disruption and complementation experiments showed that HisG_Cg is essential for histidine biosynthesis. HisG_Cg activity was noncompetitively inhibited by histidine and the α-amino group of histidine were found to play an important role for its binding to HisG_Cg. Homology-based modeling predicted that four residues (N215, L231, T235 and A270) in the C-terminal domain of HisG_Cg may affect the histidine inhibition. Mutating these residues in HisG_Cg did not cause significant change in the specific activities of the enzyme but resulted in the generation of mutant ones resistant to histidine inhibition. Our data identified that the mutant N215K/L231F/T235A resists to histidine inhibition the most with 37-fold increase in K_i value. As expected, overexpressing a hisG_Cg gene containing N215K/L231F/T235A mutations in vivo promoted histidine accumulation to a final concentration of 0.15 ± 0.01 mM. Our results demonstrated that the polarity change of electrostatic potential of mutant protein surface prevents histidine from binding to the C-terminal domain of HisG_Cg, resulting in the release of allosteric inhibition. Considering that these residues were highly conserved in ATP-PRTs from different genera of Gram-positive bacteria the mechanism by histidine inhibition as exhibited in Corynebacterium glutamicum probably represents a ubiquitously inhibitory mechanism of ATP-PRTs by histidine.     

<Emphasis Type="Italic">π</Emphasis>–<Emphasis Type="Italic">π</Emphasis> interaction between aromatic ring and copper-coordinated His81 imidazole regulates the blue copper active-site structure

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, π–π interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the π–π interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell π–π interaction. We suggest that the π–π interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Expression and simple,one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in <Emphasis Type="Italic">Escherichia coli</Emphasis> and their interaction with protoporphyrin IX

The product of human fragile histidine triad (FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap₃A) hydrolase, possesses the active site with histidine triad His-φ-His-φ-His-φφ. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His₆-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed in Escherichia coli BL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (K _m value for Ap₃A of 5.7 μM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.