Number Of Transmembrane Helices Research Articles

Sugar Transporters in Plasmodiophora brassicae: Genome-Wide Identification and Functional Verification.

Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host’s carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host.

The MscS-like channel YnaI has a gating mechanism based on flexible pore helices

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. Escherichia coli has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.

Bacterial Multidrug Efflux Proteins: A Major Mechanism of Antimicrobial Resistance

Antimicrobial resistance is a global challenge, for which the mechanisms of resistance are varied and complicated. Bacterial multidrug efflux proteins are a major mechanism of antimicrobial resistance and therefore, targets for drug development. Gram-negative bacteria are intrinsically more resistant to many antibiotics and biocides due to their cell structure and the activity of multidrug efflux proteins. These transporters actively extrude antibiotics and other xenobiotics from the cytoplasm or surrounding membranes of cells to the external environment. Based on amino acid sequence similarity, substrate specificity and the energy source used to export their substrates, there are seven major families of distinct bacterial multidrug efflux proteins: ABC, RND, MFS, SMR, MATE, PACE, AbgT. Individual proteins may be highly specialised for one compound or highly promiscuous, transporting a broad range of structurally dissimilar substrates. Protein structural organisation in a large majority of the families, including the number of transmembrane helices, has been confirmed by highresolution structure determination for at least one member. In this paper, we review the families of bacterial multidrug efflux proteins, including basic properties, energisation, structural organisation and molecular mechanism. Using representative proteins from each family, we also performed analyses of transmembrane helices, amino acid composition and distribution of charged residues. Ongoing characterisation of structure-function relationships and regulation of bacterial multidrug efflux proteins are necessary for contributing new knowledge to assist drug development and strategies that will overcome antimicrobial resistance.

Microdomain formation is a general property of bacterial membrane proteins and induces heterogeneity of diffusion patterns

BackgroundProteins within the cytoplasmic membrane display distinct localization patterns and arrangements. While multiple models exist describing the dynamics of membrane proteins, to date, there have been few systematic studies, particularly in bacteria, to evaluate how protein size, number of transmembrane domains, and temperature affect their diffusion, and if conserved localization patterns exist.ResultsWe have used fluorescence microscopy, single-molecule tracking (SMT), and computer-aided visualization methods to obtain a better understanding of the three-dimensional organization of bacterial membrane proteins, using the model bacterium Bacillus subtilis. First, we carried out a systematic study of the localization of over 200 B. subtilis membrane proteins, tagged with monomeric mVenus-YFP at their original gene locus. Their subcellular localization could be discriminated in polar, septal, patchy, and punctate patterns. Almost 20% of membrane proteins specifically localized to the cell poles, and a vast majority of all proteins localized in distinct structures, which we term microdomains. Dynamics were analyzed for selected membrane proteins, using SMT. Diffusion coefficients of the analyzed transmembrane proteins did not correlate with protein molecular weight, but correlated inversely with the number of transmembrane helices, i.e., transmembrane radius. We observed that temperature can strongly influence diffusion on the membrane, in that upon growth temperature upshift, diffusion coefficients of membrane proteins increased and still correlated inversely to the number of transmembrane domains, following the Saffman–Delbrück relation.ConclusionsThe vast majority of membrane proteins localized to distinct multimeric assemblies. Diffusion of membrane proteins can be suitably described by discriminating diffusion coefficients into two protein populations, one mobile and one immobile, the latter likely constituting microdomains. Our results show there is high heterogeneity and yet structural order in the cell membrane, and provide a roadmap for our understanding of membrane organization in prokaryotes.

Genome-wide identification of the SWEET gene family in wheat

The SWEET (sugars will eventually be exported transporter) family is a newly characterized group of sugar transporters. In plants, the key roles of SWEETs in phloem transport, nectar secretion, pollen nutrition, stress tolerance, and plant–pathogen interactions have been identified. SWEET family genes have been characterized in many plant species, but a comprehensive analysis of SWEET members has not yet been performed in wheat. Here, 59 wheat SWEETs (hereafter TaSWEETs) were identified through homology searches. Analyses of phylogenetic relationships, numbers of transmembrane helices (TMHs), gene structures, and motifs showed that TaSWEETs carrying 3–7 TMHs could be classified into four clades with 10 different types of motifs. Examination of the expression patterns of 18 SWEET genes revealed that a few are tissue-specific while most are ubiquitously expressed. In addition, the stem rust-mediated expression patterns of SWEET genes were monitored using a stem rust-susceptible cultivar, ‘Little Club’ (LC). The resulting data showed that the expression of five out of the 18 SWEETs tested was induced following inoculation. In conclusion, we provide the first comprehensive analysis of the wheat SWEET gene family. Information regarding the phylogenetic relationships, gene structures, and expression profiles of SWEET genes in different tissues and following stem rust disease inoculation will be useful in identifying the potential roles of SWEETs in specific developmental and pathogenic processes.

Prediction driven functional annotation of hypothetical proteins in the major facilitator superfamily of S. aureus NCTC 8325.

Antibiotic resistance Staphylococcus aureus strains cause several life threatening infections. New drug treatment options are needed, but are slow to develop because 50% of the S. aureus genome is hypothetical. The goal of this is to aid in the annotation of the S. aureus NCTC 8325 genome by identifying hypothetical proteins related to the Major Facilitator Superfamily (MFS). The MFS is a broad protein group with members involved in drug efflux mechanisms causing resistance. To do this, sequences for three MFS proteins with x-ray crystal structures in E. coli were PSI-BLASTed against the S. aureus NCTC 8325 genome to identify homologs. Eleven identified hypothetical protein homologs underwent BLASTP against the non-redundant NCBI database to fit homologs specific to each hypothetical protein. ExPASy characterized the physiochemical features, CDD-BLAST and Pfam identified domains, and the SOSUI server defined transmembrane helices of each hypothetical protein. Based on size (300 – 700 amino acids), number of transmembrane helices (>7), CD06174 and MFS domains in CDD-BLAST and Pfam, respectively, and close relation to well-defined homologs, SAOUHSC_00058, SAOUHSC_00078, SAOUHSC_00952, SAOUHSC_02435, SAOUHSC_02752, and ABD31642.1 are members of the MFS. Further multiple-alignment and phylogeny analyses show SAOUHSC_00058 to be a quinolone resistance protein (NorB), SAOUHSC_00058 a siderophore biosynthesis protein (SbnD), SAOUHSC_00952 a glycolipid permease (LtaA), SAOUHSC_02435 a macrolide MFS transporter, SAOUHSC_02752 a chloramphenicol resistance (DHA1), and ABD31642.1 is a Bcr/CflA family drug resistance efflux transporter. These findings provide better annotation for the existing genome, and identify proteins related to antibiotic resistance in S. aureus NCTC 8325.

Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome.

BackgroundMembrane proteins are key components in a large spectrum of diverse functions and thus account for the major proportion of the drug-targeted portion of the genome. From a structural perspective, the α-helical transmembrane proteins can be categorized into major groups based on the number of transmembrane helices and these groups are often associated with specific functions. When compared to the well-characterized seven-transmembrane containing proteins (7TM), other TM groups are less explored and in particular the 4TM group. In this study, we identify the complete 4TM complement from the latest release of the human genome and assess the 4TM structure group as a whole. We functionally characterize this dataset and evaluate the resulting groups and ubiquitous functions, and furthermore describe disease and drug target involvement.ResultsWe classified 373 proteins, which represents ~7 % of the human membrane proteome, and includes 69 more proteins than our previous estimate. We have characterized the 4TM dataset based on functional, structural, and/or evolutionary similarities. Proteins that are involved in transport activity constitute 37 % of the dataset, 23 % are receptor-related, and 13 % have enzymatic functions. Intriguingly, proteins involved in transport are more than double the 15 % of transporters in the entire human membrane proteome, which might suggest that the 4TM topological architecture is more favored for transporting molecules over other functions. Moreover, we found an interesting exception to the ubiquitous intracellular N- and C-termini localization that is found throughout the entire membrane proteome and 4TM dataset in the neurotransmitter gated ion channel families. Overall, we estimate that 58 % of the dataset has a known association to disease conditions with 19 % of the genes possibly involved in different types of cancer.ConclusionsWe provide here the most robust and updated classification of the 4TM complement of the human genome as a platform to further understand the characteristics of 4TM functions and to explore pharmacological opportunities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2592-7) contains supplementary material, which is available to authorized users.

Host Cytosolic Glutathione Sensing by a Membrane Histidine Kinase Activates the Type VI Secretion System in an Intracellular Bacterium.

Type VI secretion systems (T6SSs) are major virulence mechanisms in many Gram-negative bacteria, but the physiological signals that activate them are not well understood. The T6SS1 of Burkholderia pseudomallei is essential for pathogenesis in mammalian hosts and is only expressed when the bacterium is intracellular. We found that signals for T6SS1 activation reside in the host cytosol. Through site-directed mutagenesis and biochemical studies, we identified low molecular weight thiols, particularly glutathione, as the signal sensed by a periplasmic cysteine residue (C62) on the histidine kinase sensor VirA. Upon glutathione exposure, dimeric VirA is converted to monomers via reduction at C62. When glutathione in the host was depleted, T6SS1 expression was abrogated, and bacteria could no longer induce multinucleate giant cell formation, the hallmark of T6SS1 function. Therefore, intracellular bacteria exploit the abundance of glutathione in host cytosol as a signal for expression of virulence at the appropriate time and place.

Identification of novel sphingolipid-binding motifs in mammalian membrane proteins

Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.

Topology, glycosylation and conformational changes in the membrane domain of the vacuolar H+‐ATPase a subunit

Published topological models of the integral membrane a subunit of the vacuolar proton-translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino-terminus and carboxyl-terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V-ATPase substrate, MgATP, and the V-ATPase-specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V-ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle.

Camps 2.0: Exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/.

Possible chemotaxis in Ruminococcus albus: comparative genomics

Several comparative genomic tools were used to determine the existence of chemiotaxis in ruminal bacteria. Comparative analysis of microbial genomes (database comprehensive microbial resource (CMR)) was used to search for a specific chemiotaxis gene. Then, short sequences of Ruminococcus albus were searched in the database of the National Center for Biotechnology Information, and compared them in the and Concise Microbial Protein. To predict the number of trans-membrane helices of the putative protein, the methyl-accepting chemotaxis proteins (MCP) program was used (TMpred and TopPred). For comparisons of putative protein structures in the MCP and chemotaxis protein, Simple Modular Architecture Research Tool and protein families database were used. Short sequences of the genome of R. albus revealed the presence of chemotaxis genes that could encode a chemoreceptor (MCP) and chemotaxis proteins. Groups of R. albus chemotaxis genes may be responsible for a diverse set of signalling functions, such as the formation of biofilm, adhesion and gene regulation.

Mobility of Cytoplasmic, Membrane, and DNA-Binding Proteins in Escherichia coli

Protein mobility affects most cellular processes, such as the rates of enzymatic reactions, signal transduction, and assembly of macromolecular complexes. Despite such importance, little systematic information is available about protein diffusion inside bacterial cells. Here we combined fluorescence recovery after photobleaching with numerical modeling to analyze mobility of a set of fluorescent protein fusions in the bacterial cytoplasm, the plasma membrane, and in the nucleoid. Estimated diffusion coefficients of cytoplasmic and membrane proteins show steep dependence on the size and on the number of transmembrane helices, respectively. Protein diffusion in both compartments is thus apparently obstructed by a network of obstacles, creating the so-called molecular sieving effect. These obstructing networks themselves, however, appear to be dynamic and allow a slow and nearly size-independent movement of large proteins and complexes. The obtained dependencies of protein mobility on the molecular mass and the number of transmembrane helices can be used as a reference to predict diffusion rates of proteins in Escherichia coli. Mobility of DNA-binding proteins apparently mainly depends on their binding specificity, with FRAP recovery kinetics being slower for the highly specific TetR repressor than for the relatively nonspecific H-NS regulator.

Proteome Analysis of Rice Root Plasma Membrane and Detection of Cold Stress Responsive Proteins

To investigate the function of plant plasma membrane, proteins of rice plasma membrane were analyzed and the proteins changed by cold stress were identified. Plasma membrane proteins were purified with an aqueous two-phase partitioning method from root of rice seedlings, and activity of specific H(+)-ATPase localized in plasma membranes was measured. The plasma membrane proteins were separated by SDS-PAGE or 2D-PAGE, and analyzed with nano LC-MS/MS. The number of transmembrane helices was predicted from the amino acid sequence of annotated proteins. Functional categorization revealed that the most of proteins were associated with energy production, signal transduction, protein synthesis, cell growth/division and defense. In addition, 12 cold stress responsive proteins were identified from the plasma membrane using 2D-PAGE based proteomics method. Out of them, cold shock protein-1 was significantly decreased in plasma membrane of rice under cold stress.

Ab initio molecular-replacement phasing for symmetric helical membrane proteins.

Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 A diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

Characteristics Affecting Expression and Solubilization of Yeast Membrane Proteins

Biochemical and structural analysis of membrane proteins often critically depends on the ability to overexpress and solubilize them. To identify properties of eukaryotic membrane proteins that may be predictive of successful overexpression, we analyzed expression levels of the genomic complement of over 1000 predicted membrane proteins in a recently completed Saccharomyces cerevisiae protein expression library. We detected statistically significant positive and negative correlations between high membrane protein expression and protein properties such as size, overall hydrophobicity, number of transmembrane helices, and amino acid composition of transmembrane segments. Although expression levels of membrane and soluble proteins exhibited similar negative correlations with overall hydrophobicity, high-level membrane protein expression was positively correlated with the hydrophobicity of predicted transmembrane segments. To further characterize yeast membrane proteins as potential targets for structure determination, we tested the solubility of 122 of the highest expressed yeast membrane proteins in six commonly used detergents. Almost all the proteins tested could be solubilized using a small number of detergents. Solubility in some detergents depended on protein size, number of transmembrane segments, and hydrophobicity of predicted transmembrane segments. These results suggest that bioinformatic approaches may be capable of identifying membrane proteins that are most amenable to overexpression and detergent solubilization for structural and biochemical analyses. Bioinformatic approaches could also be used in the redesign of proteins that are not intrinsically well-adapted to such studies.

The Iron-Sulfur Cluster of the Rieske Iron-Sulfur Protein Functions as a Proton-exiting Gate in the Cytochrome bc1 Complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.

Membrane Topology of the STT3 Subunit of the Oligosaccharyl Transferase Complex

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.

Proportion of membrane proteins in proteomes of 15 single-cell organisms analyzed by the SOSUI prediction system

A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378–379). The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices. When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15–20%, irrespective of the total genome size. However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.