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ABSTRACT: The bacterial histidine permease, an ABC transporter, from Salmonella typhimurium is composed of a membrane-bound complex, HisQMP2, comprising two hydrophobic subunits (HisQ and HisM), two copies of an ATP-hydrolyzing subunit, HisP, and a soluble receptor, HisJ. We describe the purification and characterization of HisQMP2 using a 6-histidines extension at the carboxy terminus of HisP [HisQMP2(his6)]. The purification is rapid and effective, giving a seven-fold purification with a yield of 85 and 98% purity. Two procedures are described differing in the detergent used (decanoylsucrose and octylglucoside, respectively) and in the presence of phospholipid. HisQMP2(his6) has ATPase and transport activities upon reconstitution into proteoliposomes (PLS). HisQMP2(his6) has a low level ATPase activity (intrinsic activity), which is stimulated to a different extent by the receptor--liganded and unliganded. Its pH optimum is 7.8-8.0, it requires a cation for activity and it displays cooperativity for ATP. The effect of various ATP analogs was analyzed. Determination of the molecular size of HisQMP2(his6) indicates that it is a monomer. The permeability properties of two kinds of reconstituted PLS preparations are described.
Journal of Bioenergetics 05/2001; 33(2):79-92. · 2.81 Impact Factor
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ABSTRACT: The membrane-bound complex of the Salmonella typhimurium histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP, which hydrolyze ATP, thus supplying the energy for translocation. The three-dimensional structure of HisP has been resolved. Extensive evidence indicates that the HisP subunits form a dimer. We investigated the mechanism of action of such a dimer, both within the complex and in soluble form, by creating heterodimers between the wild type and mutant HisP proteins. The data strongly suggest that within the complex both subunits hydrolyze ATP and that one subunit is activated by the other. In a heterodimer containing one wild type and one hydrolysis defective subunit both hydrolysis and ligand translocation occur at half the rate of the wild type. Soluble HisP also hydrolyzes ATP if one subunit is inactive; its specific activity is identical to that of the wild type, indicating that only one of the subunits in a soluble dimer is involved in hydrolysis. We show that the activating ability varies depending on the nature of the substitution of a well conserved residue, His-211.
Journal of Biological Chemistry 10/1999; 274(38):26727-35. · 4.77 Impact Factor
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ABSTRACT: ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety of compounds across membranes of both prokaryotes and eukaryotes. The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E. coli proteins is composed of ABC transporters. Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2). Here we report the crystal structure at 1.5 A resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium. We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins. The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.
Nature 01/1999; 396(6712):703-7. · 36.28 Impact Factor
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ABSTRACT: The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP2. Purified HisP(his6) has a strong tendency to precipitate; 5 mM ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP(his6) is active as a dimer, binds ATP with a Kd value of 205 microM, and hydrolyzes it at a rate comparable to that of HisQMP2; in contrast to the latter, it does not display cooperativity for ATP. HisP(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co2+ and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP(his6) is not inhibited by vanadate but is inhibited by N-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.
Journal of Biological Chemistry 11/1997; 272(44):27745-52. · 4.77 Impact Factor
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ABSTRACT: The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium. The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation. HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand. Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released. Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex. The affinity is measured both by chemical cross-linking and co-sedimentation procedures. An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex. These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.
Journal of Biological Chemistry 07/1996; 271(24):14264-70. · 4.77 Impact Factor
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ABSTRACT: The periplasmic histidine-binding protein, HisJ, and the lysine-, arginine-, ornithine-binding protein (LAO) are receptors for histidine transport via the histidine permease of Salmonella typhimurium. The receptors have similar structures, being composed of two lobes held together by two peptide segments, with the ligand-binding site located in a cleft between the lobes. The two lobes are far apart in the unliganded structure (open conformation) and are drawn close together in the liganded structure (closed conformation). The tight binding of the ligand via protein side chains as well as the peptide backbone from both lobes stabilizes the closed conformation (Oh, B.-H., Pandit, J., Kang, C.-H., Nikaido, K., Gokcen, S., Ames, G. F.-L., and Kim, S.-H. (1993) J. Biol. Chem. 268, 11348-11353; Oh, B.-H., Kang, C.-H., De Bondt, H., Kim, S.-H., Nikaido, K., Joshi, A., and Ames, G. F.-L. (1994) J. Biol. Chem. 269, 4135-4143). In this study two conformation-specific monoclonal antibodies (mAbs) that trap the protein in the closed empty form have been characterized and used to provide evidence that HisJ can assume the closed empty form in the absence of ligand. Several pieces of evidence were provided to demonstrate that these mAbs are specific for HisJ in the closed form. Histidine improves the interaction of these mAbs with immobilized HisJ. The mAbs inhibit both the exchange and the dissociation of histidine from HisJ, indicating that they are able to trap the protein in the closed liganded form. The characterization of the epitopes of the conformation-specific mAbs shows that they include residues that are located in both lobes and that are far apart in the open form but close to each other in the closed form, so that the mAbs must be sensitive to their spatial orientation. Two mAbs that are not conformation-specific according to these criteria were also identified.
Journal of Biological Chemistry 10/1994; 269(37):23051-8. · 4.77 Impact Factor
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ABSTRACT: Bacterial periplasmic binding proteins are initial receptors in the process of active transport across cell membranes and/or chemotaxis. Among them, the histidine-binding protein (HisJ) has been extensively studied from the biochemical, physiological, and genetic points of view. The three-dimensional crystal structure of the histidine-binding protein complexed with histidine has been determined at 2.5-A resolution by the molecular replacement method using a probe structure the previously solved lysine-liganded structure of the lysine-, arginine-, ornithine-binding protein (LAO), which shares 70% sequence identity with HisJ. The structure is bi-lobate; the two lobes, one bigger than the other, are connected by two short strands and are in contact with each other (closed) enclosing the histidine. Charged, polar, and non-polar side chains, as well as the peptide backbone, are involved in tight binding of the histidine. The bound histidine is involved in eight direct hydrogen bonds, six with the bigger lobe and two with the smaller lobe, in one potential water-mediated hydrogen bond with the bigger lobe, as well as in ionic interactions. The HisJ residues surrounding the ligand are the same as the LAO residues interacting with lysine, except for residue 52 which is leucine in HisJ and phenylalanine in LAO. The Leu-52 in HisJ makes a hydrophobic interaction with the imidazole ring of histidine. Of seven mutations affecting the ligand-binding site, five are located in the ligand-binding site, one in a connecting strand, and one at the domains interface. Based on comparisons among related binding proteins, the specific interactions between the ligands and the respective binding protein residues are predicted for the glutamine-binding protein and the opines-binding protein.
Journal of Biological Chemistry 03/1994; 269(6):4135-43. · 4.77 Impact Factor
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ABSTRACT: Many proteins exhibit a large-scale movement of rigid globular domains. Among these, bacterial periplasmic binding proteins involved in substrate transport, or transport and chemotaxis, can be used as prototypes for understanding the mechanism of the movement. Such movements have been found to be associated with specific functions, such as substrate binding, catalysis, and recognition by other biomolecules. We have determined the three-dimensional structures of the lysine/arginine/ornithine-binding protein (LAO) from Salmonella typhimurium with and without lysine by x-ray crystallographic methods at 1.8- and 1.9-A resolution, respectively. The structures are composed of two lobes held together by two short connecting strands. The two lobes are far apart in the unliganded structure, but in contact with each other in the lysine-liganded structure. The large movement of the lobes is a consequence of a 52 degrees rotation of a single backbone torsion angle in the first connecting strand and of distributed smaller changes of three backbone torsion angles of the second connecting strand. The absence of contact between the lysine and the connecting strands suggests that the ligand does not induce the conformational change directly. We instead propose that the unliganded protein undergoes a dynamic change between an "open" and a "closed" conformation and that the role of the ligand is to stabilize the closed conformation. We discuss the nature of a surface area which might be recognized by the membrane-bound complex of these amino acids transport systems.
Journal of Biological Chemistry 06/1993; 268(15):11348-55. · 4.77 Impact Factor
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ABSTRACT: The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.
Journal of Biological Chemistry 11/1992; 267(29):20706-12. · 4.77 Impact Factor
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ABSTRACT: Two periplasmic binding proteins, HisJ and LAO, which are involved in histidine and arginine transport, respectively, have been crystallized. Preliminary X-ray diffraction studies of the HisJ and LAO crystals show that both belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of a = 39.26 A, b = 66.17 A, c = 88.33 A and a = 36.08 A, b = 78.34 A, c = 102.02 A, respectively. Both HisJ and LAO crystals diffract beyond 2.0 A resolution.
Journal of Molecular Biology 07/1989; 207(3):643-4. · 4.00 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 04/1989; 264(7):3998-4002. · 4.77 Impact Factor
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ABSTRACT: The promoter of nitrogen-regulated transport, argtr, has been mutationally altered in order to determine the features that are essential for its response to nitrogen availability. Deletions of all sequences upstream of position -44 or downstream of position +2 had no effect on nitrogen regulation of argTr. These deletions define a small region of 44 bp where all necessary features for nitrogen regulation are located. This region includes sequences highly homologous to the nif consensus promoter. Alteration of this particular sequence caused drastic changes in the response to changes of nitrogen availability, thus indicating that they are directly involved in regulation. This implies that the NtrC protein must also act within this small region of the promoter. The data are discussed in terms of current-hypotheses concerning nitrogen regulation. In addition, we have shown 1. that carbon regulation at this promoter must occur at a site upstream from the nitrogen promoter; 2. that nifA can replace ntrC in the regulation of argTr.
MGG - Molecular and General Genetics 01/1989; 215(1):107-17.
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ABSTRACT: The histidine permease of Salmonella typhimurium consists of four protein components, one located in the periplasm and three in the cytoplasmic membrane. Genetic evidence indicated that the periplasmic protein interacts with the membrane proteins during transport. We have utilized two different methods to demonstrate that the periplasmic protein cross-links specifically to one of the membrane components, the Q protein. Formaldehyde, a water-soluble permeant molecule was used in vivo. Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, a photoactivatable cross-linking reagent, was used in vitro in a reconstituted membrane vesicle system. Furthermore, we show that a mutant periplasmic protein, capable of binding substrate but not transporting it, is defective in cross-linking to the membrane protein, indicating this interaction to be a crucial step in the mechanism of transport.
Journal of Biological Chemistry 01/1989; 263(34):17917-20. · 4.77 Impact Factor
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ABSTRACT: We have investigated the DNA-binding ability of a nitrogen regulatory protein, the product of the ntrC gene, to several nitrogen-regulated promoters in Salmonella typhimurium. The ntrC protein is able to bind to the regulatory region (dhuA) of an operon coding for genes involved in the active transport of histidine, but not to another transport-related regulatory region, argTr. It bound to two different sites within the regulatory region of glnA (glutamine synthetase) and to one site in the regulatory region for the ntrBC operon. A consensus sequence has been derived from these four binding sites. The binding sequence displays dyad symmetry, as expected for the dimeric ntrC protein. The relationship of the binding sites to regulation of transcription initiation and termination, and to published homologies within the sequences of regulatory sites for nif genes is discussed.
The EMBO Journal 03/1985; 4(2):539-47. · 9.20 Impact Factor
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ABSTRACT: 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.
Biochimie 02/1985; 67(1):149-54. · 3.02 Impact Factor
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ABSTRACT: Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence.
Genetics 07/1983; 104(2):381-90. · 4.01 Impact Factor
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ABSTRACT: The nucleotide sequence of the entire histidine transport operon from Salmonella typhimurium has been determined and is shown to consist of four genes, hisJ, hisQ, hisM and hisP. This operon provides the only example of a binding protein-dependent transport system for which the total number of protein components is known. Determination of the amino acid compositions and sequences of these four transport proteins, together with analysis of various transport mutants, allows us to propose a molecular model for binding protein-dependent transport.
Nature 09/1982; 298(5876):723-7. · 36.28 Impact Factor
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ABSTRACT: The pattern of post-translational protein modifications involving a phosphate group was determined in the prokaryotes Salmonella typhimurium and Escherichia coli. A special two-dimensional gel electrophoretic separation was developed which utilizes acidic urea in the first dimension and neutral sodium dodecyl sulfate in the second dimension. This system allows survival and visualization of a number of proteins which are otherwise lost in systems employing basic conditions. The total number of phosphate-containing proteins thus obtained is approximately twenty. Among them are included proteins containing nucleotidylyl groups; two of these have been identified: glutamine synthetase (adenylylated) and regulatory protein PII (uridylylated). Two phosphate-containing proteins are shown to be regulated by the level of K+. The pattern of phosphorylation is shown to change with changing growth conditions and with specific mutations.
European Journal of Biochemistry 05/1981; 115(3):525-31. · 3.58 Impact Factor
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ABSTRACT: 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.
Biochemistry 09/1979; 18(19):4159-65. · 3.42 Impact Factor
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ABSTRACT: A component of high-affinity histidine transport in Salmonella typhimurium has been identified. It is a basic (pI about 9.0) membrane-bound protein, the P protein. It is shown to be coded for by the distal half of the previously described hisP gene by analysis of numerous hisP mutants, two of which exhibit P proteins with altered electrophoretic mobilities. Upon separation of the cytoplasmic (inner) from the outer membrane, it can be shown that the P protein is located in the cytoplasmic membrane. The P protein is under the same regulatory controls as histidine transport--i.e., transport operon promoter dhuA and nitrogen regulation. A wild-type cell contains about 200 molecules of P protein. As a result of this work we now divide the hisP gene into two genes: the hisP gene proper and the hisQ gene, which codes for another essential component of histidine transport, the Q protein. The P protein was shown previously by genetic analysis to interact with the periplasmic histidine-binding protein J, another essential component of histidine transport. Possible mechanism for the interaction of the J, P, and Q components in histidine transport, and of P and Q in lysine/arginine/ornithine transport, are discussed.
Proceedings of the National Academy of Sciences 12/1978; 75(11):5447-51. · 9.68 Impact Factor
