Molecular Membrane Biology最新文献

{"title":"The substrate specificity of the human ADP/ATP carrier AAC1.","authors":"John Mifsud, Stéphanie Ravaud, Eva-Maria Krammer, Chris Chipot, Edmund R S Kunji, Eva Pebay-Peyroula, Francois Dehez","doi":"10.3109/09687688.2012.745175","DOIUrl":"10.3109/09687688.2012.745175","url":null,"abstract":"<p><p>The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"160-8"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31068696","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":"Ligand binding properties of human galanin receptors.","authors":"Wiktor Jurkowski,&nbsp;Samira Yazdi,&nbsp;Arne Elofsson","doi":"10.3109/09687688.2012.750384","DOIUrl":"https://doi.org/10.3109/09687688.2012.750384","url":null,"abstract":"<p><p>The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"206-16"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.750384","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31121523","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 C-terminal cavity of the Na,K-ATPase analyzed by docking and electrophysiology.","authors":"Peter Aasted Paulsen,&nbsp;Wiktor Jurkowski,&nbsp;Rossen Apostolov,&nbsp;Erik Lindahl,&nbsp;Poul Nissen,&nbsp;Hanne Poulsen","doi":"10.3109/09687688.2012.713520","DOIUrl":"https://doi.org/10.3109/09687688.2012.713520","url":null,"abstract":"<p><p>The Na,K-ATPase is essential to all animals, since it maintains the electrochemical gradients that energize the plasma membrane. Naturally occurring inhibitors of the pump from plants have been used pharmaceutically in cardiac treatment for centuries. The inhibitors block the pump by binding on its extracellular side and thereby locking it. To explore the possibilities for designing an alternative way of targeting the pump function, we have examined the structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the pump. To cover the sample space of two residues, we first performed docking studies with the 400 possible dipeptides. For validation of the in silico predictions, pumps with 13 dipeptide sequences replacing the C-terminal YY were expressed in Xenopus laevis oocytes and examined with electrophysiology. Our data show a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C-terminus. From the dipeptides that dock the best and better than wild-type YY, it may therefore be possible to develop specific drugs targeting a previously unexplored binding pocket in the sodium pump.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"195-205"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.713520","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30851124","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":"Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using 1H-13C cross-polarisation magic-angle spinning solid-state NMR.","authors":"Simon G Patching,&nbsp;Peter J F Henderson,&nbsp;David J Sharples,&nbsp;David A Middleton","doi":"10.3109/09687688.2012.743193","DOIUrl":"https://doi.org/10.3109/09687688.2012.743193","url":null,"abstract":"<p><p>Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D(2)O with D-[(13)C(6)]glucose and (13)C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The (13)C chemical shift values for D-[(13)C(6)]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. (13)C cross-polarization build-up was measured for C1 and C2 of D-[(13)C(6)]glucose and D-[(2)H(7), (13)C(6)]glucose in GalP membranes suspended in D(2)O. The build-up curves for the deuterated substrate reflect intermolecular (1)H-(13)C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the β-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.</p>","PeriodicalId":18858,"journal":{"name":"Molecular Membrane Biology","volume":"30 2","pages":"129-37"},"PeriodicalIF":0.0,"publicationDate":"2013-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/09687688.2012.743193","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31064985","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":"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":"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":"The substrate specificity of mitochondrial carriers: mutagenesis revisited.","authors":"Magnus Monné, Ferdinando Palmieri, Edmund R S Kunji","doi":"10.3109/09687688.2012.737936","DOIUrl":"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":"","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":"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":"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}