Histidine Permease Research Articles

Revealing the Interaction of Binding Protein with ABC Transporter by Fluorescence Correlation Spectroscopy

ATP binding cassette (ABC) transporters can be found in all organism form bacteria to man. Dysfunction of these transporters is often associated with diseases, like multi drug-resistance in cancer cells. These transporters translocate substances across the cellular plasma membrane under the expense of ATP, consist of dimers of ATPase subunits known as the ATP binding cassette and of two transmembrane domains (TMDs).The histidine permease (HisQMP2) of S. typhimirium is a well characterised classic canonical ABC importer. For substrate translocation HisJ (binding protein (BP) with specificity for histidine) or LAO (BP with specificity for lysine/arginine/ornithine) are required. The BP interacts with the extracellular loops of the TMD and the binding constants for both proteins were found to be in a micro molar range. To assess the conformational dynamics of BP-TMD complexation during the catalytic cycle of substrate translocation, a Fluorescense Correlation Spectroscopy based binding assay is designed to investigate the dependency of this interaction on the catalytic state of the transporter. HisQMP2 is functionally reconstituted into large unilamellar vesicles and a monocysteine variant of the BP is chemically labelled with Alexa Fluor 488. By trapping the transporter in different steps of ATP hydrolysis, the change in diffusion rates of the fluorescent BP is supposed to reveal the dynamics of the BP association during substrate translocation.

Backbone resonance assignments for the ligand binding subunit of the histidine permease complex (HisJ) from Escherichia coli, under histidine-bound and unbound states

HisJ is a histidine binding subunit of the histidine permease, which exists in the outer membrane of Gram-negative bacteria. In order to incorporate the periplasmic histidine into the cell, HisJ captures histidine in the periplasm, and transfers the histidine to the transmembrane complex of histidine permease that is an ABC transporter. We established the backbone resonance assignments of (1)H/(13)C/(15)N-labeled HisJ from Escherichia coli, in the histidine-bound and unbound states.

Theoretical Study on the ATP Hydrolysis Mechanism of HisP Protein, the ATP-Binding Subunit of ABC Transporter

ATP binding subunit is known as a subunit of ABC transporter, providing energy through the binding of ATP with the subunit and the subsequent hydrolysis reaction of the bound ATP. In this study, density functional theory (DFT) method was used to study the ATP hydrolysis reaction in HisP protein, an ATP binding subunit of Histidine permease HisQMP2, by considering the ATP binding site, especially the � phosphate group, surrounding residues and water molecules. Based on DFT calculations, we proposed that ATP hydrolysis is initiated by the formation of Mg2þ mediated coordinate complex followed by the nucleophilic attack of a single water molecule (Water437) on the � -phosphate; the hydrolysis product ADP acts as a leaving group. The transition state structure was determined by an approximate saddle-point search. [doi:10.2320/matertrans.48.735]

New Horizon of MDR1 (P-glycoprotein) Study

MDR1 (once P-glycoprotein, now referred to as ABCB1) plays a role as a blood–brain barrier, preventing drug absorption into the brain, and is known to confer multiple drug resistance in cancer chemotherapy. MDR1 is composed of two repeated fragments, and there are six transmembrane domains (TMD) on the N-terminal of each repeat and a nucleotide (ATP) binding domain (NBD) on the C-terminal. These two repeats are dependent but cooperate as one functional molecule, with one pocket for excreting drugs. The 12 TM domains form a funnel facing the outside of cells, and NBD is in cytosol as a dimer. One NBD is composed of the Walker A, Q-loop, ABC-signature and the Walker B for phosphate binding of nucleotide. This tertiary structure of MDR1 is suggested from the structure of the NBD of histidine permease (HisP), clarified by x-ray crystallography. On the model of HisP, the NBD positions described above make a functional domain, and the same NBD structure is found on many other ABC transporters. An experiment with MDR1 gene knockout mice showed the high plasma AUC of drugs in mdr null mice [mdr1a(−/−)] and a high level in the brain, indicating that MDR1 has an efflux function (prevention of absorption) in the intestinal lumen and acts as a barrier of drug uptake in the brain, as well as has the function of urinary and biliary excretion of drugs. The transcription of MDR1 is dependent on two sites; the promoter site (− 105/− 100)(− 245/− 141) and the enhancer site (− 7864/− 7817). Autoantibody from autoimmune hepatitis patients weakly reacted with the extracellular peptide (aa314–aa328 between TM5 and 6) of MDR1 on the outside of the cell membrane, and did not react with peptides in the NBD and in the membrane-spanning region in TM5. There is an ambiguity about the function of MDR1 as GlcCer translocase.

Spelling Protein Structure

Recent sequence analysis of complete prokaryotic proteomes suggests that in early evolutionary stages proteins were rather small, of the size 25–35 amino acids. Corroborating evidence comes from protein crystal data, which indicate this size for closed loops—universal structural units of globular proteins. In the latest development we were able to derive and structurally characterize several sequence/structure prototypes apparently representing early protein units. Structurally the prototypes appear as closed loops stabilized by end-to-end van der Waals interactions. While nearly standard in size the loops are highly diverse in terms of their secondary structure. A presentation of the protein as an assembly of descendants of the prototypes, the first of its kind, is described in detail here. The sequence and structure of the ATP-binding subunit of histidine permease of S. typhimurium is shown to contain several modified copies of different prototype elements, closed loops, and, thus, can be spelled as: x-PI-x-PIV-PVI-PII-PVII-x, where PI-PVII are the prototype elements. This study sets up the basic principles for the sequence/structure prototype spelling of globular proteins.

Mechanism of ABC transporters: a molecular dynamics simulation of a well characterized nucleotide-binding subunit.

ATP-binding cassette (ABC) transporters are membrane-bound molecular pumps that form one of the largest of all protein families. Several of them are central to phenomena of biomedical interest, including cystic fibrosis and resistance to chemotherapeutic drugs. ABC transporters share a common architecture comprising two hydrophilic nucleotide-binding domains (NBDs) and two hydrophobic transmembrane domains (TMDs) that form the substrate pathway across the membrane. The conformational changes in the NBDs induced by ATP hydrolysis and the means by which they are transmitted to the TMDs to effect substrate translocation remain largely unknown. We have performed a molecular dynamics simulation of HisP, the well studied NBD of the bacterial histidine permease, to identify hinges and switches of the NBD conformational transitions and subunit-subunit interfaces. This analysis reveals that the TMDs regulate ATP hydrolysis by controlling conformational transitions of the NBD helical domains, and identifies the conformational changes and the crucial TMD:NBD interface, by which the energy of ATP hydrolysis is transmitted to the TMDs. We also define the conformational transitions of the Q-loop, a key element of the NBD mechanism, and identify pathways by which Q-loop switching is coordinated with TMD and NBD conformational changes. We propose a model for the catalytic cycle of ABC transporters that shows how substrate-binding and transport by the TMDs may be coordinated and coupled with ATP binding and hydrolysis in the NBDs.

Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake.

The ATP-binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to manQ1. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute-binding protein-dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103-104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.

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.

Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex.

The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.

Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.

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.

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.

Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.

Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.

Antibody C219 recognizes an alpha-helical epitope on P-glycoprotein.

The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 A of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic alpha-helical conformation, a secondary structure not previously observed in structures of antibody-peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported β-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.

Subunit interactions in ABC transporters: towards a functional architecture.

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.

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.

Toxicity of copper, cobalt, and nickel salts is dependent on histidine metabolism in the yeast Saccharomyces cerevisiae.

The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeast Saccharomyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium. Each of the his1-his7 mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His(+) strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His(-) strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H(+)-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni.

Molecular interactions in ribose transport: the binding protein module symmetrically associates with the homodimeric membrane transporter.

The Escherichia coli high-affinity ribose transporter is composed of the periplasmic ribose-binding protein (RBP or RbsB), the membrane component (RbsC) and the ATP-binding protein (RbsA). In order to dissect the molecular interactions initiating the transport process, RbsC suppressors for transport-defective rbsB mutations were isolated. These suppressors are localized in two regions of RbsC, which are allele-specific to N- or C-terminal domain mutations of RBP, suggesting that there are two distinct regions of RbsC, each interacting with one of the two domains of RBP. To demonstrate that these two regions provide a homodimeric binding surface for RBP we constructed a dimeric rbsC in which two genes are joined tandemly from head to tail with the addition of a linker. The dimeric RbsC protein is stable and functional in growth and ribose uptake. By exploiting the allele specificity between the domain-specific mutations and their suppressors, we generated all mutation-suppressor combinations in a single rbsB plus the dimeric rbsC genes. Their phenotypes are consistent with the proposal that the binding protein module interacts symmetrically with homodimeric RbsC. The mode of association proposed here for the ribose transport components could be extended to other ABC transporters with similar structural organizations.

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.