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.

A high-resolution method for two-dimensional separation of membrane proteins is described. It involves a nondiscriminating solubilization of a membrane preparation with sodium dodecyl sulfate, followed by electrophoresis in the first dimension according to charge (by isoelectric focusing). The electrophoresis in the second dimension is in the presence of sodium dodecyl sulfate, thus separating proteins on the basis of molecular weight. Electrophoresis in the first dimension is either on a thin slab gel, or on a small-diameter tube; electrophoresis in the second dimension is on a thin slab gel. Up to 100 mug of protein can be analyzed. The two-dimensional system is a modification of the one recently described by O'Farrell (1975). About 150 different proteins can be visualized in Escherichia coli or Salmonella typhimurim cell envelopes; examples of differences between mutant and wild-type strains are presented. The method is applicable also to membrane preparations from other sources: a two-dimensional separation of plasma membrane proteins from HeLa cells is presented.

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.

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, HisQMP 2 . 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 K d value of 205 M, and hydrolyzes it at a rate comparable to that of HisQMP 2 ; 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 Co 2؉ and Mn 2؉being more effective than Mg 2؉ 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.

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.