Molecular Membrane Biology最新文献

{"title":"Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG.","authors":"Hamidreza Vaziri,&nbsp;Stephen A Baldwin,&nbsp;Jocelyn M Baldwin,&nbsp;David G Adams,&nbsp;James D Young,&nbsp;Vincent L G Postis","doi":"10.3109/09687688.2012.748939","DOIUrl":"https://doi.org/10.3109/09687688.2012.748939","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"114-28"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.748939","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31136586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli.","authors":"Nadia Gul,&nbsp;Bert Poolman","doi":"10.3109/09687688.2012.754060","DOIUrl":"https://doi.org/10.3109/09687688.2012.754060","url":null,"abstract":"<p><p>The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"138-48"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.754060","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31129923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT.","authors":"Katie J Simmons,&nbsp;Kamil Gotfryd,&nbsp;Christian B Billesbølle,&nbsp;Claus J Loland,&nbsp;Ulrik Gether,&nbsp;Colin W G Fishwick,&nbsp;A Peter Johnson","doi":"10.3109/09687688.2012.710341","DOIUrl":"https://doi.org/10.3109/09687688.2012.710341","url":null,"abstract":"<p><p>Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"184-94"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.710341","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30847721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Foreword for EDICT special edition, volume 2.","authors":"","doi":"10.3109/09687688.2013.761918","DOIUrl":"https://doi.org/10.3109/09687688.2013.761918","url":null,"abstract":"A central aim of the EDICT consortium was to obtain high resolution structures of a range of integral membrane proteins as a route to structure-aided drug design. The first issue of our special EDICT edition described efforts to express and isolate a wide range of membrane proteins in a number of different expression systems together with selected structureactivity studies. In this second issue we present further structure-activity studies, especially those detailing how structural information can be translated into platforms for the discovery of novel drugs. When no experimental structures are available, researchers depend upon homology models for their studies. Vaziri et al describe the validation of one such model of a major facilitator superfamily (MFS) transporter, using cysteine mutagenesis. In contrast, Patching et al utilized solid state NMR and sample deuteration to explore the low affinity binding site of the E. coli MFS transporter, GalP, demonstrating the usefulness of this technique for investigation of binding site dynamics. Gul et al explored the osmoregulatory properties of the ProUABC transporter, comprised of ProX, ProY and ProW, from E. coli following isolation and reconstitution into proteoliposomes. The findings reveal novel insights into the regulation of this interesting protein complex. Monne et al review the mutagenic data currently available as a means of shedding light on substrate specificity of mitochondrial carriers. This is complementedby the functional characterization of the human mitochondrial ADP/ATP carrier AAC1 described by Mifsud et al. Wohri et al have used isothermal calorimetry to investigate the thermodynamic changes in the pentameric glycine receptor upon ligand binding. In the latter part of the project, members of the EDICT consortium made significant progress in the identification and characterization of novel drug molecules, for a number of different membrane protein targets. Simmons et al used a structurebased virtual high-throughput screening (vHTS) technique initially developed for soluble proteins to identify inhibitors of LeuT and achieved a very impressive hit rate of 45%. Paulsen et al built on earlier extensive structural analysis to confirm the importance of the two C-terminal residues of the Na, K-ATPase in sodium binding. They also describe a combined computer docking and experimental approach to the preliminary characterization of dipeptides with potential to act as novel Na, K-ATPase inhibitors. Jurkowski et al describe a computational approach to the identification of key regions of the galanin receptors with roles in ligand interaction. Finally, Infed et al describe an approach involving modification of a known small molecule to generate novel inhibitors of multi-drug resistance transporters. Taken together, 19 manuscripts published in two special issues of Molecular Membrane Biology provide an overview of almost all of the research expertise required to take a protein from gene to ear","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"113"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2013.761918","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31178951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The substrate specificity of mitochondrial carriers: mutagenesis revisited.","authors":"Magnus Monné,&nbsp;Ferdinando Palmieri,&nbsp;Edmund R S Kunji","doi":"10.3109/09687688.2012.737936","DOIUrl":"https://doi.org/10.3109/09687688.2012.737936","url":null,"abstract":"<p><p>Mitochondrial carriers transport inorganic ions, nucleotides, amino acids, keto acids and cofactors across the mitochondrial inner membrane. Structurally they consist of three domains, each containing two transmembrane α-helices linked by a short α-helix and loop. The substrate binds to three major contact points in the central cavity. The class of substrate (e.g., adenine nucleotides) is determined by contact point II on transmembrane α-helix H4 and the type of substrate within the class (e.g., ADP, coenzyme A) by contact point I in H2, whereas contact point III on H6 is most usually a positively charged residue, irrespective of the type or class. Two salt bridge networks, consisting of conserved and symmetric residues, are located on the matrix and cytoplasmic side of the cavity. These residues are part of the gates that are involved in opening and closing of the carrier during the transport cycle, exposing the central substrate binding site to either side of the membrane in an alternating way. Here we revisit the plethora of mutagenesis data that have been collected over the last two decades to see if the residues in the proposed binding site and salt bridge networks are indeed important for function. The analysis shows that the major contact points of the substrate binding site are indeed crucial for function and in defining the specificity. The matrix salt bridge network is more critical for function than the cytoplasmic salt bridge network in agreement with its central position, but neither is likely to be involved in substrate recognition directly.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"149-59"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.737936","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31023305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry.","authors":"Annemarie Beate Wöhri,&nbsp;Per Hillertz,&nbsp;Per-Olof Eriksson,&nbsp;Johan Meuller,&nbsp;Niek Dekker,&nbsp;Arjan Snijder","doi":"10.3109/09687688.2012.696733","DOIUrl":"https://doi.org/10.3109/09687688.2012.696733","url":null,"abstract":"<p><p>In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K(D) of 138 ± 55 nM, a ΔH of -9708 ± 1195 cal/mol and a ΔS of -1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K(D) of 3.2 ± 0.8 μM, a ΔH of -2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"169-83"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.696733","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30716733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of the inhibition potential of zosuquidar derivatives on selected bacterial and fungal ABC transporters.","authors":"Nacera Infed,&nbsp;Sander H J Smits,&nbsp;Torsten Dittrich,&nbsp;Manfred Braun,&nbsp;Arnold J M Driessen,&nbsp;Nils Hanekop,&nbsp;Lutz Schmitt","doi":"10.3109/09687688.2012.758876","DOIUrl":"https://doi.org/10.3109/09687688.2012.758876","url":null,"abstract":"<p><p>The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC(50) values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"217-27"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.758876","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31193325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Specific aquaporins increase the ammonia tolerance of a Saccharomyces cerevisiae mep1-3 fps1 deletion strain.","authors":"Dawid Krenc,&nbsp;Binghua Wu,&nbsp;Eric Beitz","doi":"10.3109/09687688.2012.733976","DOIUrl":"https://doi.org/10.3109/09687688.2012.733976","url":null,"abstract":"<p><p>Abstract Aquaporins (AQPs) are channel proteins which facilitate the bidirectional membrane permeation of small neutral molecules such as water and glycerol. A convenient way to characterize their permeability is by growth of transformed Saccharomyces cerevisiae deletion strains on nutrient-limited substrates. We selected a yeast strain deficient in its endogenous ammonium transporters Mep1-3 and aquaglyceroporin Fps1 in order to study the ammonia permeability of heterologously expressed AQPs. Surprisingly, AQP-expression improved yeast growth at high, not low, concentrations of unprotonated ammonia. At neutral or mildly alkaline pH, ammonia concentrations above 10 μM decreased the growth rate and especially the number of yeast cell duplications, but did not affect the lag phase. AQP-expression raised the threshold to about 100 μM. The exchange of ammonium ions for amino acids or urea did not completely abolish this effect. AQPs capable of rescuing growth had a selectivity filter wide enough to permit passage of molecules larger than water but smaller than glycerol. It appears that the endogenous aquaglyceroporin Fps1 may, under alkaline conditions, be beneficial to yeast by facilitating the membrane permeation of an as yet unidentified molecule other than glycerol.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 1","pages":"43-51"},"PeriodicalIF":0.0,"publicationDate":"2013-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.733976","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30995490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans.","authors":"James Leung,&nbsp;Alexander D Cameron,&nbsp;George Diallinas,&nbsp;Bernadette Byrne","doi":"10.3109/09687688.2012.690572","DOIUrl":"https://doi.org/10.3109/09687688.2012.690572","url":null,"abstract":"<p><p>Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 1","pages":"32-42"},"PeriodicalIF":0.0,"publicationDate":"2013-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.690572","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30687685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.","authors":"Juho Kellosalo,&nbsp;Tommi Kajander,&nbsp;Riina Honkanen,&nbsp;Adrian Goldman","doi":"10.3109/09687688.2012.712162","DOIUrl":"https://doi.org/10.3109/09687688.2012.712162","url":null,"abstract":"<p><p>Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 1","pages":"64-74"},"PeriodicalIF":0.0,"publicationDate":"2013-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.712162","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30824536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}