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G F Ames

Publications and source records attributed to G F Ames.

At least 19 recordsLinked to original sources
PubMed2001

Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease.

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.

ATP-Binding Cassette Transporters↗
PubMed1999

One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease.

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.

ATP-Binding Cassette Transporters↗
PubMed1999

Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter, the histidine permease.

The membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703-707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters.

ATP-Binding Cassette Transporters↗
PubMed1999

Both lobes of the soluble receptor of the periplasmic histidine permease, an ABC transporter (traffic ATPase), interact with the membrane-bound complex. Effect of different ligands and consequences for the mechanism of action.

The histidine permease of Salmonella typhimurium is an ABC transporter (traffic ATPase). The liganded soluble receptor, the histidine-binding protein HisJ, interacts with the membrane-bound complex HisQMP2 and stimulates its ATPase activity, which results in histidine translocation. In this study, we utilized HisJ proteins with mutations in either of the two lobes and wild type HisJ liganded with different substrates to show that each lobe carries an interaction site and that both lobes are involved in inducing (stimulating) the ATPase activity. We suggest that the spatial relationship between the lobes is one of the factors recognized by the membrane-bound complex in dictating the efficiency of the induction signal and of translocation. Several of the key residues involved have been identified. In addition, using constitutive ATPase mutants, we show that the binding protein provides some additional essential function(s) in translocation that is independent of the stimulation of ATP hydrolysis, and one possible mechanism is proposed, which includes the notion that liganded HisJ has different optimal conformations for signaling and for translocation.

ATP-Binding Cassette Transporters↗
PubMed1998

Crystal structure of the ATP-binding subunit of an ABC transporter.

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.

ATP-Binding Cassette Transporters↗
PubMed1998

In vitro disassembly and reassembly of an ABC transporter, the histidine permease.

The membrane-bound complex of the Salmonella typhimurium periplasmic 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. The complex hydrolyzes ATP upon induction of the activity by the liganded soluble receptor, the periplasmic histidine-binding protein, HisJ. Here we take advantage of the modular organization of this system to show that the nucleotide-binding component can be stripped off the integral membrane components, HisQ and HisM. The complex can be reconstituted by using the HisP-depleted membranes containing HisQ and HisM and pure soluble HisP. We show that HisP has high affinity for the HisP-depleted complex, HisQM, and that two HisP molecules are recruited independently of each other for each HisQM unit. The in vitro reassembled complex has entirely normal properties, responding to HisJ and ATPase inhibitors with the same characteristics as the original complex and in contrast to those of soluble HisP. These results show that HisP is absolutely required for ATP hydrolysis, that HisQM cannot hydrolyze ATP, that HisP depends on HisQM to relay the inducing signal from the soluble receptor, HisJ, and that HisQM regulates the ATPase activity of HisP. We also show that HisP changes conformation upon exposure to phospholipids.

ATP-Binding Cassette Transporters↗
PubMed1998

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.

Arginine↗
PubMed1997

Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter), the histidine permease of Salmonella typhimurium. Solubility, dimerization, and ATPase activity.

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.

ATP-Binding Cassette Transporters↗
PubMed1997

Characterization of the adenosine triphosphatase activity of the periplasmic histidine permease, a traffic ATPase (ABC transporter).

The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and eukaryotic transporters. The histidine permease of Salmonella typhimurium is composed of a membrane-bound complex (HisQMP2) containing four subunits, and of a soluble receptor, the histidine-binding protein (HisJ). Transport is energized by ATP. In this article the ATPase activity of HisQMP2 has been characterized, using a novel assay that is independent of transport. The assay uses Mg2+ ions to permeabilize membrane vesicles or proteoliposomes, thus allowing access of ATP to both sides of the bilayer. HisQMP2 displays a low level of intrinsic ATPase activity in the absence of HisJ; unliganded HisJ stimulates the activity and liganded HisJ stimulates to an even higher level. All three levels of activity display positive cooperativity for ATP with a Hill coefficient of 2 and a K0. 5 value of 0.6 mM. The activity has been characterized with respect to pH, salt, phospholipids, substrate, and inhibitor specificity. Free histidine has no effect. The activity is inhibited by orthovanadate, but not by N-ethylmaleimide, bafilomycin A1, or ouabain. Several nucleotide analogs, ADP, 5'-adenylyl-beta, gamma-imidodiphosphate, adenosine 5'-(beta,gammaimino)triphosphate, and adenosine 5'-O-(3-thio)triphosphate, inhibit the activity. Unliganded HisJ does not compete with liganded HisJ for the stimulation of the ATPase activity of HisQMP2.

ATP-Binding Cassette Transporters↗
PubMed1997

Characterization of transport through the periplasmic histidine permease using proteoliposomes reconstituted by dialysis.

The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and various eukaryotic transporters. The histidine permease of Salmonella typhimurium and Escherichia coli is composed of a membrane-bound complex containing four subunits and of a soluble receptor, the substrate-binding protein (HisJ), and is energized by ATP. The permease was previously reconstituted into proteoliposomes by a detergent dilution method (1). Here we extensively characterize the properties of this permease after reconstitution into proteoliposomes by dialysis and encapsulation of ATP or other reagents by freeze-thawing. We show that histidine transport depends entirely on both ATP and liganded HisJ, with apparent Km values of 8 mM and 8 microM, respectively, and is affected by pH, temperature, and salt concentration. Transport is irreversible and accumulation reaches a plateau at which point transport ceases. The permease is inhibited by ADP and by high concentrations of internal histidine. The inhibition by histidine implies that the membrane-bound complex HisQ/M/P carries a substrate-binding site. The reconstituted permease activity corresponds to about 40-70% turnover rate of the in vivo rate of transport.

ATP-Binding Cassette Transporters↗
PubMed1996

Ligand-dependent conformational plasticity of the periplasmic histidine-binding protein HisJ. Involvement in transport specificity.

The periplasmic histidine permease of Salmonella typhimurium is composed of a membrane-bound complex and a soluble histidine-binding protein (the periplasmic receptor), HisJ. Liganded receptor interacts with the membrane-bound complex, inducing ATP hydrolysis and substrate translocation. Preliminary evidence had shown a lack of direct correlation between the affinity of HisJ for a ligand and translocation efficiency, suggesting that the precise form of the receptor is important in determining its interaction with the membrane-bound complex. We have investigated the nature of the conformations assumed by HisJ upon binding a variety of ligands by tryptophan fluorescence enhancement, reaction with a closed form-specific monoclonal antibody, and changes in UV absorption spectra. It is demonstrated that although HisJ binds all the ligands and undergoes a conformational change, it assumes measurably different conformations. We also show that the interaction between HisJ and the membrane-bound complex depends on the nature of the ligand. Transport specificity appears to be defined, at least in part, by the conformation of the bound receptor, manifested either by the effect of a given ligand on the closed structure per se, or by the effect of ligand association on the equilibrium constant relating the open and the closed liganded forms.

Antibodies, Monoclonal↗
PubMed1996

Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.

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.

ATP-Binding Cassette Transporters↗
PubMed1996

D-histidine utilization in Salmonella typhimurium is controlled by the leucine-responsive regulatory protein (Lrp).

A new class of D-histidine-utilizing mutants which carry mutations in the gene encoding the leucine-responsive regulatory protein (Lrp) has been identified in Salmonella typhimurium. The lrp mutations arise as suppressors of mutations in the genes encoding the histidine permease which drastically decrease the level of histidine transport activity. However, the suppressor effect is not exerted by elevating the level of the permease. Rather, the properties of the suppressor mutants are consistent with the notion that the parent permease mutants transport D-histidine at a low level and that in the suppressor mutants D-histidine is utilized effectively through elevated levels of racemization. The enzymatic activity of D-alanine dehydrogenase (Dad) is shown to be elevated in the suppressor mutants and is a possible pathway of D-histidine utilization. The suppressor mutations are located in the helix-turn-helix region of Lrp.

ATP-Binding Cassette Transporters↗
PubMed1995

Structure/function analysis of the periplasmic histidine-binding protein. Mutations decreasing ligand binding alter the properties of the conformational change and of the closed form.

The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium. Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation). The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation. Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand. In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L. (1994) J. Biol. Chem. 269, 23051-23058). Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form. T121A and G119R are similar to Y14H, but assume a normal closed liganded form. S72P binds the ligand to the open form, but does not assume a recognizable closed form. S92F is defective in the ability to undergo conformational change and to stabilize the closed form. All other mutant proteins appear to fall within one of these four categories. The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein. We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals.

Antibodies, Bacterial↗
PubMed1994

Structural basis for multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein.

The substrate-binding site of a protein with multiple specificity should satisfy geometric and energetic complementarity for several different substrates. The structural basis of the multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) was investigated by determining and analyzing the structures of the protein unliganded and liganded with each of the three high-affinity ligands (L-lysine, L-arginine, and L-ornithine) and with one low-affinity ligand (L-histidine). The geometric complementarity is achieved primarily by virtue of the large size of the ligand-binding site which can accommodate the maximum common volume of the four ligands plus three water molecules. The optimization of energetic complementarity is achieved by the relocation of protein-bound water molecules and by the movement of the Asp-11 side chain. The structure of the LAO-histidine complex indicates that the 30-fold reduced affinity of the protein for histidine is primarily due to unavailability of one ionic interaction of the histidine side chain with the protein which is present in the other three complexes.

Arginine↗
PubMed1994

The histidine-binding protein undergoes conformational changes in the absence of ligand as analyzed with conformation-specific monoclonal antibodies.

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.

Antibodies, Monoclonal↗
PubMed1994

The bacterial periplasmic histidine-binding protein. structure/function analysis of the ligand-binding site and comparison with related proteins.

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.

Bacterial Proteins↗