Giovanna Ferro-Luzzi Ames

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 ofcompounds 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 Å 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.

Repetitive extragenic palindromic sequences: A major component of the bacterial genome

We describe a remarkably conserved nucleotide sequence, the many copies of which may occupy up to 1% of the genomes of E. coli and S. typhimurium. This sequence, the REP (repetitive extragenic palindromic) sequence, is about 35 nucleotides long, includes an inverted repeat, and can occur singly or in multiple adjacent copies. A possible role for the REP sequences in regulation of gene expression has been thoroughly investigated. While the REP sequences do not appear to modulate differential gene expression within an operon, they can affect the expression of both upstream and downstream genes to a small extent, probably by affecting the rate of mRNA degradation. Possible roles for the REP sequence in mRNA degradation, chromosome structure, and recombination are discussed.

Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR ☆

We have devised a strategy to extend the use of the polymerase chain reaction (PCR) to amplify double-stranded DNA when sequence information is available only at one extremity. The only information required is a short stretch of sequence used to design a gene-specific primer, which is then used in combination with a second generic vector primer at the unknown end. The primers are used in a PCR reaction after ligating the unknown end to a generic vector. Restriction, ligation, amplification and sequencing of the products can be achieved within three days. This method eliminates the laborious steps of shotgun cloning, colony screening and culturing of cells. We have used this method to take two contiguous steps beyond the histidine transport operon in Salmonella typhimurium. We also demonstrate the usefulness of this technique to do chromosome walking in the absence of any restriction data.

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 ofSalmonella 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 aK 0.5 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 byN-ethylmaleimide, bafilomycin A1, or ouabain. Several nucleotide analogs, ADP, 5′-adenylyl-β,γ-imidodiphosphate, adenosine 5′-(β,γimino)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.

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 aK d value of 205 μm, 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 byN-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.

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 theSalmonella 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.

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 μM, 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.

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.

Genome Walking by Single Specific Primer–Polymerase Chain Reaction

Publisher Summary This chapter describes that the polymerase chain reaction (PCR) technique is used for the selective amplification of DNA fragments that can be used for various purposes. The only requirement for amplification is that the sequence information at the extremities of the DNA fragment to be amplified be known. However, this requirement poses a major limitation on the use of the PCR in the amplification of unknown regions. The basic principle of the single specific primer-polymerase chain reaction (SSP-PCR) procedure is schematically described in the chapter: (1) the DNA is digested, preferably using two restriction enzymes, (2) the unknown end of the DNA is ligated to a suitable oligomer of known sequence, long enough to be used as a primer for the PCR, and (3) the ligation reaction mixture is subjected to PCR amplification using a specific primer annealing to the known sequence and a generic oligomer annealing to the unknown end. The SSP-PCR technique has also been used successfully in eukaryotic systems.

Structure and mechanism of bacterial periplasmic transport systems

Bacterial periplasmic transport systems are complex, multicomponent permeases, present in Gram-negative bacteria. Many such permeases have been analyzed to various levels of detail. A generalized picture has emerged indicating that their overall structure consists of four proteins, one of which is a soluble periplasmic protein that binds the substrate and the other three are membrane bound. The liganded periplasmic protein interacts with the membrane components, which presumably form a complex, and which by a series of conformational changes allow the formation of an entry pathway for the substrate. The two extreme alternatives for such pathway involve either the formation of a nonspecific hydrophilic pore or the development of a ligand-binding site(s) on the membrane-bound complex. One of the membrane-bound components from each system constitutes a family of highly homologous proteins containing sequence domains characteristic of nucleotide-binding sites. Indeed, in several cases, they have been shown to bind ATP, which is thus postulated to be involved in the energy-coupling mechanism. Interestingly, eukaryotic proteins homologous to this family of proteins have been identified (mammalianmdr genes and Drosophilawhite locus), thus indicating that they perform a universal function, presumably related to energy coupling in membrane-related processes. The mechanism of energy coupling in periplasmic permeases is discussed.