Functional effects of intramembranous proline substitutions in the staphylococcal multidrug transporter QacA

The QacA multidrug transporter is encoded on Staphylococcus aureus multidrug resistance plasmids and confers broad-range antimicrobial resistance to more than 30 monovalent and bivalent lipophilic, cationic compounds from at least 12 different chemical classes. QacA contains 10 proline residues predicted to be within transmembrane regions, several of which are conserved in related export proteins. Proline residues are classically known as helix-breakers and are highly represented within the transmembrane helices of membrane transport proteins, where they can mediate the formation of structures essential for protein stability and transport function. The importance of these 10 intramembranous proline residues for QacA-mediated transport function was determined by examining the functional effect of substituting these residues with glycine, alanine or serine. Several proline-substituted QacA mutants failed to confer high-level resistance to selected QacA substrates. However, no single proline mutation, including those at conserved positions, significantly disrupted QacA protein expression or QacA-mediated resistance to all representative substrates, suggesting that these residues are not essential for the formation of structures requisite to the QacA substrate transport mechanism.     

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose‐dependently inhibited by addition of D‐glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose‐mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N‐acetyl‐glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose‐mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose‐negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose‐mediated inhibition of F. johnsoniae colony spreading.

     

Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG

Abstract

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H⁺ Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H⁺ Symporter (OHS) family. Sequence alignment of these distantly related proteins (～ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.     

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT_So, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT_So represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.     

Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM

Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl-β-D-maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl-β-D-maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.     

Functional interactions between the subunits of the lactose transporter from Streptococcus thermophilus

Although the quaternary state has been assessed in detail for only a few members of the major facilitator superfamily (MFS), it is clear that multiple oligomeric states are represented within the MFS. One of its members, the lactose transporter LacS from Streptococcus thermophilus assumes a dimeric structure in the membrane and in vitro analysis showed functional interactions between both subunits when proton motive force ((Delta)p)-driven transport was assayed. To study the interactions in further detail, a covalent dimer was constructed consisting of in tandem fused LacS subunits. These covalent dimers, composed of active and completely inactive subunits, were expressed in Escherichia coli, and initial rates of (Delta)p-driven lactose uptake and lactose counterflow were determined. We now show that also in vivo, both subunits interact functionally; that is, partial complementation of the inactive subunit was observed for both transport modes. Thus, both subunits interact functionally in (Delta)p-driven uptake and in counterflow transport. In addition, analysis of in tandem fused LacS subunits containing one regulatory LacS-IIA domain showed that regulation is primarily an intramolecular event.     

Purification and properties of the Escherichia coli nucleoside transporter NupG,a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K_m values of ～20–30 μM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1′-¹³C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly α-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Projection structure of the bacterial oxalate transporter OxlT at 3.4A resolution

OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 A resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 microm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4A, and electron diffraction patterns show spots to about 3.2 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22(1)2(1) symmetry group. However, the unit cell dimensions of 102.7A x 67.3 A are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3A x 79.0 A. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.     

Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE

EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14_A and E14_B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Structural insights into substrate recognition in proton‐dependent oligopeptide transporters

Short‐chain peptides are transported across membranes through promiscuous proton‐dependent oligopeptide transporters (POTs)—a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepT_So2), which was crystallized in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand‐binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.     

Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in Saccharomyces cerevisiae

Genes encoding transporters for heterologous siderophores have been identified in Saccharomyces cerevisiae, of which SIT1, TAF1, and ENB1 encode the transporters for ferrioxamines, ferric triacetylfusarinine C and ferric enterobactin, respectively. In the present communication we have shown that a further gene encoding a member of the major facilitator superfamily, ARN1 (YHL040c), is involved in the transport of a specific class of ferrichromes, possessing anhydromevalonyl residues linked to N(delta)-ornithine (ARN). Ferrirubin and ferrirhodin, which both are produced by filamentous fungi, are the most common representatives of this class of ferrichromes. A strain possessing a disruption in the ARN1 gene was unable to transport ferrirubin, ferrirhodin and also ferrichrome A, indicating that the encoded transporter recognizes anhydromevalonyl and the structurally-related methylglutaconyl side-chains surrounding the iron center. Ferrichromes possessing short-chain ornithine-N(delta)-acetyl residues such as ferrichrome, ferricrocin and ferrichrysin, were excluded by the Arn1 transporter. Substitution of the iron-surrounding N-acyl chains of ferrichromes by propionyl residues had no effect, whereas substitution by butyryl residues led to recognition by the Arn1 transporter. This would indicate that a chain length of four C-atoms is sufficient to allow binding. Using different asperchromes (B1, D1) we also found that a minimal number of two anhydromevalonyl residues is sufficient for recognition by Arn1p. Contrary to the iron-surrounding N-acyl residues, the peptide backbone of ferrichromes was not an important determinant for the Arn1 transporter.     

Atomic resolution structure of the E. coli YajR transporter YAM domain

YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration.     

Conformational changes in the multidrug transporter EmrE associated with substrate binding

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP(+)) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP(+) binding; the data showed that one TPP(+) molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP(+) produced well ordered two-dimensional crystals. Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7A resolution. An average native EmrE projection structure was calculated from the c222 and p222(1) crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP(+) bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPP(+) bound at the monomer-monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane alpha-helix.     

New insights into the isopenicillin N transport in Penicillium chrysogenum

In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRed protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane.     

Molecular analysis of the multidrug transporter

The multidrug resistance gene product, P-glycoprotein or the multidrug transporter, confers multidrug resistance to cancer cells by maintaining intracellular levels of cytotoxic agents below a killing threshold. P-glycoprotein is located within the plasma membrane and is thought to act as an energy-dependent drug efflux pump. The multidrug transporter represents a member of the ATP-binding cassette superfamily of transporters (or traffic ATPases) and is composed of two highly homologous halves, each of which harbors a hydrophobic transmembrane domain and a hydrophilic ATP-binding fold. This review focuses on various biochemical and molecular genetic approaches used to analyze the structure, function, and mechanism of action of the multidrug transporter, whose most intriguing feature is its ability to interact with a large number of structurally and functionally different amphiphilic compounds. These studies have underscored the complexity of this membrane protein which has recently been suggested to assume alternative topological and quaternary structures, and to serve multiple functions both as a transporter and as a channel. With respect to the multidrug transporter activity of P-glycoprotein, progress has been made towards the elucidation of essential amino acid residues and/or polypeptide regions. Furthermore, the drug-stimulatable ATPase activity of P-glycoprotein has been established. The mechanism of drug transport by P-glycoprotein, however, is still unknown and its physiological role remains a matter of speculation.     

Structural basis for polyspecificity in the POT family of proton‐coupled oligopeptide transporters

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di‐ and tripeptide‐bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co‐crystal structures combined with functional studies reveal that biochemically distinct peptide‐binding sites likely operate within the POT/PTR family of proton‐coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.     

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate

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