Unfolding free energy of a two-domain transmembrane sugar transport protein.

Understanding how an amino acid sequence folds into a functional, three-dimensional structure has proved to be a formidable challenge in biological research, especially for transmembrane proteins with multiple alpha helical domains. Mechanistic folding studies on helical membrane proteins have been limited to unusually stable, single domain proteins such as bacteriorhodopsin. Here, we extend such work to flexible, multidomain proteins and one of the most widespread membrane transporter families, the major facilitator superfamily, thus showing that more complex membrane proteins can be successfully refolded to recover native substrate binding. We determine the unfolding free energy of the two-domain, Escherichia coli galactose transporter, GalP; a bacterial homologue of human glucose transporters. GalP is reversibly unfolded by urea. Urea causes loss of substrate binding and a significant reduction in alpha helical content. Full recovery of helical structure and substrate binding occurs in dodecylmaltoside micelles, and the unfolding free energy can be determined. A linear dependence of this free energy on urea concentration allows the free energy of unfolding in the absence of urea to be determined as +2.5 kcal·mol(-1). Urea has often been found to be a poor denaturant for transmembrane helical structures. We attribute the denaturation of GalP helices by urea to the dynamic nature of the transporter structure allowing denaturant access via the substrate binding pocket, as well as to helical structure that extends beyond the membrane. This study gives insight into the final, critical folding step involving recovery of ligand binding for a multidomain membrane transporter.

Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1.

The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

Crystallization of the hydantoin transporter Mhp1 from Microbacterium liquefaciens.

The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P2(1)2(1)2(1), unit-cell parameters a = 79.7, b = 101.1, c = 113.8 A). A complete data set has been collected from a single crystal to a resolution of 2.85 A with 64 741 independent observations (94% complete) and an R(merge) of 0.12. Further experimental phasing methods are under way.

Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter.

The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.

WITHDRAWN: Characterisation of the Intact Sensor Kinase VicK from Enterococcus faecalis: Direct Evidence for YycH as a Potent Modulating Signal in vitro.

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily.

A genomic strategy for the overexpression of bacterial multidrug and antibiotic resistance membrane efflux proteins in Escherichia coli is described. Expression is amplified so that the encoded proteins from a range of Gram-positive and Gram-negative bacteria comprise 5% to 35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGS(His)(6)-tag or a Strep-tag at the C terminus. These tags facilitate the purification of the overexpressed proteins in milligram quantities for structural studies. The strategy is illustrated for the bicyclomycin resistance efflux protein, Bcr, of E. coli.

Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism.

gamma-Glutamyltranspeptidase (GGT) is a periplasmic enzyme of Helicobacter pylori implicated in its pathogenesis towards mammalian cells. We have cloned and expressed the H. pylori strain 26695 recombinant GGT protein in Escherichia coli and purified it to homogeneity. The purified protein exhibited hydrolysis activity with very high affinities for glutamine and glutathione shown by apparent K(m) values lower than 1 muM. H. pylori cells were unable to take up extracellular glutamine and glutathione directly. Instead, these substances were hydrolysed to glutamate by the action of GGT outside the cells. The glutamate produced was then transported by a Na(+)-dependent reaction into H. pylori cells, where it was mainly incorporated into the TCA cycle and partially utilized as a substrate for glutamine synthesis. These observations show that one of the principle physiological functions of H. pylori GGT is to enable H. pylori cells to utilize extracellular glutamine and glutathione as a source of glutamate. As glutamine and glutathione are important nutrients for maintenance of healthy gastrointestinal tissue, their depletion by the GGT enzyme is hypothesized to account for the damaging of mammalian cells and the pathophysiology of H. pylori.

Microbial drug efflux proteins of the major facilitator superfamily.

Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.

The gusBC genes of Escherichia coli encode a glucuronide transport system.

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

Collection and characterisation of bacterial membrane proteins.

A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.