Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.     

A knowledge-based scale for the analysis and prediction of buried and exposed faces of transmembrane domain proteins

MOTIVATION: The dearth of structural data on alpha-helical membrane proteins (MPs) has hampered thus far the development of reliable knowledge-based potentials that can be used for automatic prediction of transmembrane (TM) protein structure. While algorithms for identifying TM segments are available, modeling of the TM domains of alpha-helical MPs involves assembling the segments into a bundle. This requires the correct assignment of the buried and lipid-exposed faces of the TM domains. RESULTS: A recent increase in the number of crystal structures of alpha-helical MPs has enabled an analysis of the lipid-exposed surfaces and the interiors of such molecules on the basis of structure, rather than sequence alone. Together with a conservation criterion that is based on previous observations that conserved residues are mostly found in the interior of MPs, the bias of certain residue types to be preferably buried or exposed is proposed as a criterion for predicting the lipid-exposed and interior faces of TMs. Applications to known structures demonstrates 80% accuracy of this prediction algorithm. AVAILABILITY: The algorithm used for the predictions is implemented in the ProperTM Web server (http://icb.med.cornell.edu/services/propertm/start).     

kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction

Modeling of integral membrane proteins and the prediction of their functional sites requires the identification of transmembrane (TM) segments and the determination of their angular orientations. Hydrophobicity scales predict accurately the location of TM helices, but are less accurate in computing angular disposition. Estimating lipid-exposure propensities of the residues from statistics of solved membrane protein structures has the disadvantage of relying on relatively few proteins. As an alternative, we propose here a scale of knowledge-based Propensities for Residue Orientation in Transmembrane segments (kPROT), derived from the analysis of more than 5000 non-redundant protein sequences. We assume that residues that tend to be exposed to the membrane are more frequent in TM segments of single-span proteins, while residues that prefer to be buried in the transmembrane bundle interior are present mainly in multi-span TMs. The kPROT value for each residue is thus defined as the logarithm of the ratio of its proportions in single and multiple TM spans. The scale is refined further by defining it for three discrete sections of the TM segment; namely, extracellular, central, and intracellular. The capacity of the kPROT scale to predict angular helical orientation was compared to that of alternative methods in a benchmark test, using a diversity of multi-span alpha-helical transmembrane proteins with a solved 3D structure. kPROT yielded an average angular error of 41 degrees, significantly lower than that of alternative scales (62 degrees -68 degrees ). The new scale thus provides a useful general tool for modeling and prediction of functional residues in membrane proteins. A WWW server (http://bioinfo.weizmann.ac.il/kPROT) is available for automatic helix orientation prediction with kPROT.     

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM alpha-helical bundle. We have shown previously that the topology of the alpha-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM alpha-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric alpha-helical bundle model.     

MicroRNA靶向病毒携带的microRNA模拟靶序列对病毒的抑制作用

Micro RNA模拟靶序列(target mimic,TM)通过竞争性结合miRNA,从而干扰miRNA对靶标m RNA的调控.我们前期工作发现:以黄瓜花叶病毒(Cucumber mosaic virus,CMV)作为载体在植物体内表达TM序列有效地抑制了miRNA的活性或稳定性,从而消减了miRNA对靶基因的调控.但是,miRNA与CMV携带的TM序列的结合在一定程度上抑制了病毒的积累.研究分析了miRNA靶向病毒携带的TM序列对病毒抑制作用的内在原因.a.通过RNA印迹分析CMV携带不同miRNA TM序列对病毒积累的影响,进一步明确miRNA靶向病毒携带的TM序列对病毒的抑制作用;b.利用GFP作为报告基因,通过荧光显微镜、蛋白质印迹以及RNA印迹分析TM序列对重组病毒积累的影响;c.以GFP作为报告基因,利用荧光显微镜观察和免疫印迹方法分析模拟靶序列对GFP翻译的影响;d.利用CMV病毒的反式复制系统分析miRNA模拟靶序列对病毒负链RNA合成的影响.结果表明,多种植物内源的miRNA靶向CMV基因组携带的miRNA TM序列,在不同程度上抑制了病毒的积累,miRNA与其TM序列的结合抑制GFP蛋白的翻译和负链的合成.植物内源的miRNA通过与病毒基因组携带的miRNA模拟靶序列结合,通过抑制病毒蛋白的翻译以及病毒负链RNA的合成,从而降低了病毒的积累水平.基于该论文的研究结果有可能建立一种抗病毒的新方法.     

Sequence similarity as a predictor of the transmembrane topology of membrane-intrinsic subunits of bacterial respiratory chain enzymes

Integral membrane proteins usually have a predominantly alpha-helical secondary structure in which transmembrane segments are connected by membrane-extrinsic loops. Although a number of membrane protein structures have been reported in recent years, in most cases transmembrane topologies are initially predicted using a variety of theoretical techniques, including hydropathy analyses and the "positive inside" rule. We have explored the use of plots of the distribution of sequence similarity within families of membrane proteins comprising homeomorphic domains as a new method for the prediction/verification of the orientation of transmembrane topology models within certain families of multimeric respiratory chain enzymes. Within such proteins, analyses of sequence similarity can: i) identify heme and/or quinol binding sites; ii) identify potential electron-transfer conduits to/from prosthetic groups; and iii) locate regions defining potential subunit-subunit interactions. We mined emerging bioinformatic data for sequences of 11 families of membrane-intrinsic proteins that are part of multimeric respiratory chain complexes that also have membrane-extrinsic subunits. The sequences of each family were then aligned and the resultant alignments converted into a graphical format recording an empirical measure of the sequence similarity plotted versus residue position. In each case, this plot was compared to the predicted transmembrane topology. With one exception, there is a strong correlation between the existence     

Characterization of peptides corresponding to the seven transmembrane domains of human adenosine A2a receptor

Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily of seven-helix transmembrane (TM) proteins. To test general models for membrane-protein folding and to identify specific features of folding and assembly for this representative member of an important and poorly understood class of proteins, we synthesized peptides corresponding to its seven TM domains. We assessed the ability of the peptides to insert into micelles and vesicles and measured secondary structure for each peptide in aqueous and membrane-mimetic environments. CD spectra indicate that each of the seven TM peptides form thermally stable, independent alpha-helical structures in both micelles and vesicles. The helical content of the peptides depends on the nature of the membrane-mimetic environment. Four of the peptides (TM3, TM4, TM5, and TM7) exhibit very high-helical structure, near the predicted maximum for their TM segments. The TM1 peptide also adopts relatively high alpha-helical structures. In contrast, two of peptides, TM2 and TM6, display low alpha helicity. Similarly, the ability of the peptides to insert into membrane-mimetic environments, assayed by intrinsic tryptophan fluorescence and fluorescence quenching, varied markedly. Most peptides exhibit higher alpha helicity in anionic sodium dodecyl sulfate than in neutral dodecyl-beta-D-maltoside micelles, and TM2 was disordered in zwiterionic DMPC but was alpha-helical in negatively charged DMPC/DMPG vesicles. These findings strongly suggest that electrostatic interactions between lipids and peptides control the insertion of the peptides and may be involved in membrane-protein-folding events. The measured helical content of these TM domains does not correlate with the predicted helicity based on amino acid sequence, pointing out that, while hydrophobic interactions can be a major determinant for folding of TM peptides, other factors, such as electrostatic interactions or helix-helix interactions, may play significant roles for specific TM domains. Our results represent a comprehensive analysis of helical propensities for a human GPCR and support models for membrane-protein folding in which interactions between TM domains are required for proper insertion and folding of some TM helix domains. The tendency of some peptides to self-associate, especially in aqueous environments, underscores the need to prevent improper interactions during folding and refolding of membrane proteins in vivo and in vitro.     

The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.     

Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human mu-opioid receptor. The fragment ('TM2-3'), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts approximately 50% alpha-helical structure in this environment, with the remainder consisting of disordered and/or beta-structure. This is consistent with the assumption of an alpha-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the mu-opioid receptor and, possibly, other G-protein-coupled receptors as well.     

PRALINETM: a strategy for improved multiple alignment of transmembrane proteins

MOTIVATION: Membrane-bound proteins are a special class of proteins. The regions that insert into the cell-membrane have a profoundly different hydrophobicity pattern compared with soluble proteins. Multiple alignment techniques use scoring schemes tailored for sequences of soluble proteins and are therefore in principle not optimal to align membrane-bound proteins. RESULTS: Transmembrane (TM) regions in protein sequences can be reliably recognized using state-of-the-art sequence prediction techniques. Furthermore, membrane-specific scoring matrices are available. We have developed a new alignment method, called PRALINETM, which integrates these two features to enhance multiple sequence alignment. We tested our algorithm on the TM alignment benchmark set by Bahr et al. (2001), and showed that the quality of TM alignments can be significantly improved compared with the quality produced by a standard multiple alignment technique. The results clearly indicate that the incorporation of these new elements into current state-of-the-art alignment methods is crucial for optimizing the alignment of TM proteins. AVAILABILITY: A webserver is available at http://www.ibi.vu.nl/programs/pralinewww.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Model of a putative pore: the pentameric alpha-helical bundle of SARS coronavirus E protein in lipid bilayers

The coronavirus responsible for the severe acute respiratory syndrome contains a small envelope protein, E, with putative involvement in host apoptosis and virus morphogenesis. To perform these functions, it has been suggested that protein E can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a TM pore. Indeed, in a recent study we reported that the alpha-helical putative transmembrane domain of E protein (ETM) forms several SDS-resistant TM interactions: a dimer, a trimer, and two pentameric forms. Further, these interactions were found to be evolutionarily conserved. Herein, we have studied multiple isotopically labeled ETM peptides reconstituted in model lipid bilayers, using the orientational parameters derived from infrared dichroic data. We show that the topology of ETM is consistent with a regular TM alpha-helix. Further, the orientational parameters obtained unequivocally correspond to a homopentameric model, by comparison with previous predictions. We have independently confirmed that the full polypeptide of E protein can also aggregate as pentamers after expression in Escherichia coli. This interaction must be stabilized, at least partially, at the TM domain. The model we report for this pentameric alpha-helical bundle may explain some of the permabilizing properties of protein E, and should be the basis of mutagenesis efforts in future functional studies.     

Transmembrane organization of the Bacillus subtilis chemoreceptor McpB deduced by cysteine disulfide crosslinking

The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.     

Charged single alpha-helix: a versatile protein structural motif

A few highly charged natural peptide sequences were recently suggested to form stable alpha-helical structures in water. In this article we show that these sequences represent a novel structural motif called "charged single alpha-helix" (CSAH). To obtain reliable candidate CSAH motifs, we developed two conceptually different computational methods capable of scanning large databases: SCAN4CSAH is based on sequence features characteristic for salt bridge stabilized single alpha-helices, whereas FT_CHARGE applies Fourier transformation to charges along sequences. Using the consensus of the two approaches, a remarkable number of proteins were found to contain putative CSAH domains. Recombinant fragments (50-60 residues) corresponding to selected hits obtained by both methods (myosin 6, Golgi resident protein GCP60, and M4K4 protein kinase) were produced and shown by circular dichroism spectroscopy to adopt largely alpha-helical structure in water. CSAH segments differ substantially both from coiled-coil and intrinsically disordered proteins, despite the fact that current prediction methods recognize them as either or both. Analysis of the proteins containing CSAH motif revealed possible functional roles of the corresponding segments. The suggested main functional features include the formation of relatively rigid spacer/connector segments between functional domains as in caldesmon, extension of the lever arm in myosin motors and mediation of transient interactions by promoting dimerization in a range of proteins.     

Incorporation of transmembrane peptides from the vacuolar H(+)-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy

Peptides were designed that are based on candidate transmembrane sequences of the V o-sector from the vacuolar H (+)-ATPase of Saccharomyces cerevisiae. Spin-label EPR studies of lipid-protein interactions were used to characterize the state of oligomerization, and polarized IR spectroscopy was used to determine the secondary structure and orientation, of these peptides in lipid bilayer membranes. Peptides corresponding to the second and fourth transmembrane domains (TM2 and TM4) of proteolipid subunit c (Vma3p) and of the putative seventh transmembrane domain (TM7) of subunit a (Vph1p) are wholly, or predominantly, alpha-helical in membranes of dioleoyl phosphatidylcholine. All three peptides self-assemble into oligomers of different sizes, in which the helices are differently inclined with respect to the membrane normal. The coassembly of rotor (Vma3p TM4) and stator (Vph1p TM7) peptides, which respectively contain the glutamate and arginine residues essential to proton transport by the rotary ATPase mechanism, is demonstrated from changes in the lipid interaction stoichiometry and helix orientation. Concanamycin, a potent V-ATPase inhibitor, and a 5-(2-indolyl)-2,4-pentadienoyl inhibitor that exhibits selectivity for the osteoclast subtype, interact with the membrane-incorporated Vma3p TM4 peptide, as evidenced by changes in helix orientation; concanamycin additionally interacts with Vph1p TM7, suggesting that both stator and rotor elements contribute to the inhibitor site within the membrane. Comparison of the peptide behavior in lipid bilayers is made with membranous subunit c assemblies of the 16-kDa proteolipid from Nephrops norvegicus, which can substitute functionally for Vma3p in S. cerevisiae.     

Energy-conformation studies of frequency of beta-turns in tetrapeptide sequences

The optimized energies of seven beta-bends, repeating C5 and C7, and right- and left-handed alpha-helical conformations for each of eight tetrapeptides have been computed using empirical methods. Eight tetramers were selected: four helix-forming sequences with hydrophobic residues such as Val, Leu, Ile and Trp, and four helix-breaking sequences with hydrophilic residues such as Asp, Asn and Ser, as determined by their frequency of occurrence in beta turns in proteins. Analysis of the optimized conformations with energies less than or equal to 2.1 kcal/mol from the absolute minimum energy conformer for each tetramer reveals a correlation between low-energy conformations and those predicted from observed protein structures. These results show that energy calculations on small peptide fragments may be usefulin predicting protein structure.     

Membrane proteins: the 'Wild West' of structural biology

Historically, the task of determining the structure of membrane proteins has been hindered by experimental difficulties associated with their lipid-embedded domains. Here, we provide an overview of recently developed experimental and predictive tools that are changing our view of this largely unexplored territory - the 'Wild West' of structural biology. Crystallography, single-particle methods and atomic force microscopy are being used to study huge membrane proteins with increasing detail. Solid-state nuclear magnetic resonance strategies provide orientational constraints for structure determination of transmembrane (TM) alpha-helices and accurate measurements of intramolecular distances, even in very complex systems. Longer distance constraints are determined by site-directed spin-labelling electron paramagnetic resonance, but current labelling strategies still constitute some limitation. Other methods, such as site-specific infrared dichroism, enable orientational analysis of TM alpha-helices in aligned bilayers and, combined with novel computational and predictive tools that use evolutionary conservation data, are being used to analyze TM alpha-helical bundles.     

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.     

Proteome-wide functional classification and identification of prokaryotic transmembrane proteins by transmembrane topology similarity comparison

We propose a new method for classifying and identifying transmembrane (TM) protein functions in proteome-scale by applying a single-linkage clustering method based on TM topology similarity, which is calculated simply from comparing the lengths of loop regions. In this study, we focused on 87 prokaryotic TM proteomes consisting of 31 proteobacteria, 22 gram-positive bacteria, 19 other bacteria, and 15 archaea. Prior to performing the clustering, we first categorized individual TM protein sequences as "known," "putative" (similar to "known" sequences), or "unknown" by using the homology search and the sequence similarity comparison against SWISS-PROT to assess the current status of the functional annotation of the TM proteomes based on sequence similarity only. More than three-quarters, that is, 75.7% of the TM protein sequences are functionally "unknown," with only 3.8% and 20.5% of them being classified as "known" and "putative," respectively. Using our clustering approach based on TM topology similarity, we succeeded in increasing the rate of TM protein sequences functionally classified and identified from 24.3% to 60.9%. Obtained clusters correspond well to functional superfamilies or families, and the functional classification and identification are successfully achieved by this approach. For example, in an obtained cluster of TM proteins with six TM segments, 109 sequences out of 119 sequences annotated as "ATP-binding cassette transporter" are properly included and 122 "unknown" sequences are also contained.