RESUMO
Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
Assuntos
Microscopia Crioeletrônica/métodos , Cicloparafinas/química , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/isolamento & purificação , Polímeros/química , Microscopia Crioeletrônica/instrumentaçãoRESUMO
Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential γ-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid both in vitro and when overexpressed in E. coli Additionally, transport was greatly reduced in the presence of ß-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport ß-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Mycobacterium smegmatis/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Sódio/metabolismo , Ácido gama-Aminobutírico/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Regulação Bacteriana da Expressão Gênica , Metabolômica , Simulação de Acoplamento Molecular , Transportadores de Ânions Orgânicos/genética , Filogenia , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/genéticaRESUMO
Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.
RESUMO
Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.
RESUMO
Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.
Assuntos
Maleatos/química , Lipídeos de Membrana/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Poliestirenos/química , Microscopia Crioeletrônica/métodos , Detergentes , Escherichia coli/química , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química , Gotículas Lipídicas/química , Espectrometria de Massas/métodos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Polímeros/químicaRESUMO
A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.
Assuntos
Cromatografia de Afinidade/métodos , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Animais , Escherichia coli/crescimento & desenvolvimento , Vetores Genéticos , Humanos , Células Sf9RESUMO
Glutamate transporters play important roles in bacteria, archaea and eukaryotes. Their function in the mammalian central nervous system is essential for preventing excitotoxicity, and their dysregulation is implicated in many diseases, such as epilepsy and Alzheimer's. Elucidating their transport mechanism would further the understanding of these transporters and promote drug design as they provide compelling targets for understanding the pathophysiology of diseases and may have a direct role in the treatment of conditions involving glutamate excitotoxicity. This review outlines the insights into the transport cycle, uncoupled chloride conductance and modulation, as well as identifying areas that require further investigation.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Archaea/metabolismo , Sistema X-AG de Transporte de Aminoácidos/química , Ácido Aspártico/metabolismo , Sítios de Ligação , Cloretos/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Potássio/metabolismo , Conformação Proteica , Sódio/metabolismoRESUMO
Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.
Assuntos
Proteínas de Membrana/química , Nanotecnologia , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Western Blotting , Lipídeos/química , Espectrometria de Massas , Microscopia Eletrônica , Estrutura Quaternária de ProteínaRESUMO
The proteobacterial antimicrobial compound efflux (PACE) family of transport proteins was only recently described. PACE family transport proteins can confer resistance to a range of biocides used as disinfectants and antiseptics, and are encoded by many important Gram-negative human pathogens. However, we are only just beginning to appreciate the range of functions and the mechanism(s) of transport operating in these proteins. Genes encoding PACE family proteins are typically conserved in the core genomes of bacterial species rather than on recently acquired mobile genetic elements, suggesting that they confer important core functions in addition to biocide resistance. Three-dimensional structural information is not yet available for PACE family proteins. However, PACE proteins have several very highly conserved amino acid sequence motifs that are likely to be important for substrate transport. PACE proteins also display strong amino acid sequence conservation between their N and C-terminal halves, suggesting that they evolved by duplication of an ancestral protein comprised of two transmembrane helices. In light of their drug resistance functions in Gram-negative pathogens, PACE proteins should be the subject of detailed future investigation.
Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Desinfetantes/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteobactérias/química , Proteobactérias/genética , Proteobactérias/metabolismoRESUMO
The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.
Assuntos
Microscopia Crioeletrônica/métodos , Bicamadas Lipídicas/química , Maleatos/química , Proteínas de Membrana/química , Poliestirenos/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/ultraestrutura , Conformação Proteica , Multimerização Proteica , Proteolipídeos/química , Proteolipídeos/ultraestruturaRESUMO
Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19-20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the "common microbial binding site" on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.
Assuntos
Borrelia burgdorferi/metabolismo , Cristalização , Cristalografia por Raios X , Conformação ProteicaRESUMO
Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification.
RESUMO
Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.
Assuntos
Proteínas de Membrana/isolamento & purificação , Membranas Artificiais , Métodos , Bactérias/metabolismo , Eucariotos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologiaRESUMO
Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Pyrococcus horikoshii/metabolismo , Ácido Aspártico/metabolismo , Transporte Biológico , Conformação Proteica , Especificidade por SubstratoRESUMO
Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (â¼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.
Assuntos
Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/química , Maleatos/química , Lipídeos de Membrana/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Poliestirenos/química , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , SolubilidadeRESUMO
Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.
Assuntos
Proteínas de Bactérias/metabolismo , Cátions Bivalentes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metais/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Transporte de Nucleosídeos/química , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Peroxisomes are organelles that perform diverse metabolic functions in different organisms, but a common function is ß-oxidation of a variety of long chain aliphatic, branched, and aromatic carboxylic acids. Import of substrates into peroxisomes for ß-oxidation is mediated by ATP binding cassette (ABC) transporter proteins of subfamily D, which includes the human adrenoleukodystropy protein (ALDP) defective in X-linked adrenoleukodystrophy (X-ALD). Whether substrates are transported as CoA esters or free acids has been a matter of debate. Using COMATOSE (CTS), a plant representative of the ABCD family, we demonstrate that there is a functional and physical interaction between the ABC transporter and the peroxisomal long chain acyl-CoA synthetases (LACS)6 and -7. We expressed recombinant CTS in insect cells and showed that membranes from infected cells possess fatty acyl-CoA thioesterase activity, which is stimulated by ATP. A mutant, in which Serine 810 is replaced by asparagine (S810N) is defective in fatty acid degradation in vivo, retains ATPase activity but has strongly reduced thioesterase activity, providing strong evidence for the biological relevance of this activity. Thus, CTS, and most likely the other ABCD family members, represent rare examples of polytopic membrane proteins with an intrinsic additional enzymatic function that may regulate the entry of substrates into the ß-oxidation pathway. The cleavage of CoA raises questions about the side of the membrane where this occurs and this is discussed in the context of the peroxisomal coenzyme A (CoA) budget.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Tioléster Hidrolases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Acil Coenzima A/metabolismo , Adenosina Trifosfatases , Substituição de Aminoácidos , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico Ativo , Coenzima A Ligases/metabolismo , Proteínas de Transporte de Ácido Graxo/genética , Humanos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Peroxissomos/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tioléster Hidrolases/genéticaRESUMO
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 (â¼ 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.