Structure Of Transmembrane Domain Research Articles

The key residue within the second extracellular loop of human EP3 involved in selectively turning down PGE2- and retaining PGE1-mediated signaling in live cells

Key residues and binding mechanisms of PGE1 and PGE2 on prostanoid receptors are poorly understood due to the lack of X-ray structures for the receptors. We constructed a human EP3 (hEP3) model through integrative homology modeling using the X-ray structure of the β2-adrenergic receptor transmembrane domain and NMR structures of the thromboxane A2 receptor extracellular loops. PGE1 and PGE2 docking into the hEP3 model showed differing configurations within the extracellular ligand recognition site. While PGE2 could form possible binding contact with S211, PGE1 is unable to form similar contacts. Therefore, S211 could be the critical residue for PGE2 recognition, but is not a significant for PGE1. This prediction was confirmed using HEK293 cells transfected with hEP3 S211L cDNA. The S211L cells lost PGE2 binding and signaling. Interestingly, the S211L cells retained PGE1-mediated signaling. It indicates that S211 within the second extracellular loop is a key residue involved in turning down PGE2 signaling. Our study provided information that S211L within EP3 is the key residue to distinguish PGE1 and PGE2 binding to mediate diverse biological functions at the initial recognition step. The S211L mutant could be used as a model for studying the binding mechanism and signaling pathway specifically mediated by PGE1.

Structure and activation of the TSH receptor transmembrane domain

PurposeThe thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves’ disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD–M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown.MethodsWe have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available.ResultsMost conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs.ConclusionsInsight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves’ disease.

X-ray structures define human P2X(3) receptor gating cycle and antagonist action.

P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the 'cytoplasmic cap', which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes.

The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein–coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain–binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.

The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance.

The influenza M2 protein is the target of the amantadine family of antiviral drugs, and its transmembrane (TM) domain structure and dynamics have been extensively studied. However, little is known about the structure of the highly conserved N-terminal ectodomain, which contains epitopes targeted by influenza vaccines. In this study, we synthesized an M2 construct containing the N-terminal ectodomain and the TM domain, to understand the site-specific conformation and dynamics of the ectodomain and to investigate the effect of the ectodomain on the TM structure. We incorporated (13)C- and (15)N-labeled residues into both domains and measured their chemical shifts and line widths using solid-state nuclear magnetic resonance. The data indicate that the entire ectodomain is unstructured and dynamic, but the motion is slower for residues closer to the TM domain. (13)C line shapes indicate that this ecto-TM construct undergoes fast uniaxial rotational diffusion, like the isolated TM peptide, but drug binding increases the motional rates of the TM helix while slowing the local motion of the ectodomain residues that are close to the TM domain. Moreover, (13)C and (15)N chemical shifts indicate that the ectodomain shifts the conformational equilibria of the TM residues toward the drug-bound state even in the absence of amantadine, thus providing a molecular structural basis for the lower inhibitory concentration of full-length M2 compared to that of the ectodomain-truncated M2. We propose that this conformational selection may result from electrostatic repulsion between negatively charged ectodomain residues in the tetrameric protein. Together with the recent study of the M2 cytoplasmic domain, these results show that intrinsically disordered extramembrane domains in membrane proteins can regulate the functionally relevant conformation and dynamics of the structurally ordered TM domains.

Understanding single-pass transmembrane receptor signaling from a structural viewpoint-what are we missing?

Single-pass transmembrane receptors are involved in essential processes of both physiological and pathological nature and represent more than 1300 proteins in the human genome. Despite the high biological relevance of these receptors, the mechanisms of the signal transductions they facilitate are incompletely understood. One major obstacle is the lack of structures of the transmembrane domains that connect the extracellular ligand-binding domains to the intracellular signaling platforms. Over a period of almost 20years since the first structure was reported, only 21 of these receptors have become represented by a transmembrane domain structure. This scarceness stands in strong contrast to the significance of these transmembrane α-helices for receptor functionality. In this review, we explore the properties and qualities of the current set of structures, as well as the methodological difficulties associated with their characterization and the challenges left to be overcome. Without an increased and focused effort to bring this class of proteins on par with the remaining membrane protein field, a serious lag in their biological understanding looms. Design of pharmaceutical agents, prediction of mutational affects in relation to disease, and deciphering of functional mechanisms require high-resolution structural information, especially when dealing with a domain carrying so much functionality in so few residues.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane

The partially de-N-acetylated poly-β-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513-807). The structure reveals that PgaA forms a 16-strand transmembrane β-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the β-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane.

The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes

The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978–2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC.

Improved 3D Structure Prediction of Beta-Barrel Membrane Proteins by using Evolutionary Coupling Constraints, Reduced State Space and an Empirical Potential Function

Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and bacterial virulence. In addition, beta-barrel membrane proteins increasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank and computational methods can help to understand their biophysical principles. We have developed a novel computational method to predict the 3D structure of beta-barrel membrane proteins using evolutionary coupling (EC) constraints and a reduced state space. Combined with an empirical potential function, we can successfully predict strand register at >80% accuracy for a set of 49 non-homologous proteins with known structures. This is a significant improvement from previous results using EC alone (44%) [1] and using empirical potential function alone (73%) [2]. Our method is general and can be applied to genome-wide structural prediction.[1] Sikander Hayat, Chris Sander, Debora S. Marks, and Arne Elofsson. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences. PNAS 2015 112 (17) 5413-5418.[2] Hammad Naveed, Yun Xu, Ronald Jackups, Jr., and Jie Liang. Predicting Three-Dimensional Structures of Transmembrane Domains of β-Barrel Membrane Proteins. Journal of the American Chemical Society 2012 134 (3), 1775-1781.

Structural Characterization of the Full Length CrgA Protein Found in Mycobacterium Tuberculosis

Mycobacterium Tuberculosis, or Mtb is a pathogen of Tuberculosis. Mtb has a unique stage called latency, when Mtb temporarily shuts down cell division. During this stage, antibiotics are not able to kill the bacteria, so a treatment has to continue for 6 months. Today multidrug resistant Mtb even necessitates a much longer treatment, up to 2 years. The development of new drugs is critically important.CrgA plays a pivotal role in the Mtb cell division apparatus, the divisome. It is known to recruit and interact with other Mtb proteins associated with cell division. Recently, the transmembrane domain structure of CrgA has been determined using solid state NMR spectroscopy. The goal of this work is to characterize a high resolution structure of CrgA and then characterize CrgA protein-protein complexes with a view toward drug development. In addition, the N-terminus and the interhelical loop need to be characterized to identify additional protein binding sites.The full length CrgA is overexpressed in E. coli yielding 40mg per liter of culture. The purified protein is reconstituted in the synthetic lipid bilayers to induce the native structure. To define the secondary structure of the N-terminus and the interhelical loop, two different 2D magic angle spinning solid state NMR experiments are performed with specific amino acid labeling strategies. DARR experiments provide the information about 13C chemical shifts and 13C-13C correlations and both 13C & 15N isotropic chemical shifts are obtained from NCA experiments. Recently, it has been recognized that CrgA functions as a dimer based on bacterial two hybrid assays. From an analysis of the membrane exposed glycine residues, binding interfacial sites for the dimer-dimer interface have been suggested as well as possible sites for interaction for its protein recruitment partners.

Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN(-) binding defined by EPR-based hybrid method.

Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN−, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.

A 6-bp deletion in exon 8 and two mutations in introns of TYRP1 are associated with blond coat color in Liangshan pigs

Melanocortin receptor 1 (MC1R), Agouti signaling protein (ASIP), and Tyrosinase-related protein 1 (TYRP1) are reported critical genes that regulate pheomelanin and eumelanin synthesis in mammals. Liangshan pig is a special Chinese indigenous pig breed with two completely different coat colors, solid black and blond. In this study, we detected polymorphisms of the above three genes and assessed the relationships between the variations and coat color phenotypes in Liangshan pigs. The findings revealed that the blond phenotype of Liangshan pig was related to dominant mutations in TYRP1, but not related to mutations in MC1R or ASIP. We found three closely linked mutations in TYRP1, g.8406G>A in intron 4, g.11100A>G in intron 5, and g.17599_17604del in exon 8, that were completely associated with blond coat color in Liangshan pigs. Further analysis revealed that a 6-bp deletion mutation resulted in deletion of Met and Gly residues at positions 495 and 496 in TYRP1 protein, and altered the structure of transmembrane domain of TYRP1. Together, our findings indicated that these three mutations in TYRP1 cause the blond phenotype in Liangshan pigs.

The Membrane Mimetic Affects the Spatial Structure and Mobility of EGFR Transmembrane and Juxtamembrane Domains.

The epidermal growth factor receptor (EGFR) is one of the most extensively studied receptor tyrosine kinases, as it is involved in a wide range of cellular processes and severe diseases. Recent works reveal that the single-helix transmembrane domains and cytoplasmic juxtamembrane regions play an important role in the receptor activation process. Here we present the results of our investigation of the spatial structure and mobility of the EGFR transmembrane domain and juxtamembrane regions in various membranelike environments, which shed light on the effects of the membrane physical properties and composition on the behavior of the juxtamembrane domain.

Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy.

Despite the biological and pharmaceutical significance of membrane proteins, their tertiary structures constitute less than 3% of known structures. One of the major obstacles for initiating structural studies of membrane proteins by NMR spectroscopy is the generation of high amounts of isotope-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures of single-pass TMDs, which are difficult to solve by other means.

Structural and phylogenetic analysis of α-glucosidase protein in insects

Abstractα-Glucosidases (αglus) (EC 3.2.1.20) have been detected in a wide range of plants, animals and microorganisms and are described by their ability to catalyze the hydrolysis of 1,4-α-glucosidic linkages, releasing α-glucose. In this study, 96 αglu protein sequences from 33 organisms species including different insects species, plants (including thale cress and rice), mammals (including human and mouse) and Mycobacterium tuberculosis as a bacterium were aligned. Sequences were analyzed by computational tools to predict the protein properties, such as molecular mass, isoelectric point, signal peptide, conserved motifs, transmembrane domain and secondary structure. Drosophila melanogaster (GenBank Accession No.: NP 610382) was chosen as one of the representatives of insects for further analyses. The tertiary structure of representative samples were acquired using the tertiary structure of oligo-1,6-glucosidase from Bacillus cereus (Protein Data Bank code: 1UOK) as a template by Phyre2 server. Protein structure analysis revealed there is a high identity among insects and other organisms. There were some similar functional domains between D. melanogaster and M. tuberculosis. The modeled αglu has a typical spatial structure in insects and exhibits a high similarity with other organisms, especially Arabidopsis thaliana. Phylogenetic analysis indicated that D. melanogaster αglu has a close relationship with other αglus from different insect families. According to these results, αglu in insects should be evolved from a common ancestor.

The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region

The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

Ensemble-based virtual screening for cannabinoid-like potentiators of the human glycine receptor α1 for the treatment of pain.

The human glycine receptors (hGlyRs) are chloride-selective ion channels that mediate inhibitory neurotransmission in the brain stem and spinal cord. They are also targets for compounds of potential use in analgesic therapies. Here, we develop a strategy to discover analgesic drugs via structure-based virtual screening based on the recently published NMR structure of the hGlyR-α1 transmembrane domain (PDB ID: 2M6I ) and the critical role of residue S296 in hGlyR-α1 potentiation by Δ(9)-tetrahydrocannabinol (THC). We screened 1549 FDA-approved drugs in the DrugBank database on an ensemble of 180 hGlyR-α1 structures generated from molecular dynamics simulations of the NMR structure of the hGlyR-α1 transmembrane domain in different lipid environments. Thirteen hit compounds from the screening were selected for functional validation in Xenopus laevis oocytes expressing hGlyR-α1. Only one compound showed no potentiation effects; seven potentiated hGlyR-α1 at a level greater than THC at 1 μM. Our virtual screening protocol is generally applicable to drug targets with lipid-facing binding sites.

Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer, Chilo suppressalis

Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in cells, with an EC50 value of 18.7±5.3nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca2+ release from the hemocytes of C. suppressalis.

Positive Modulators of Glycine Receptors with Analgesic Potential Identified by Virtual Screening

Human glycine receptors (hGlyRs) are important targets for neuroactive drugs, including for analgesic therapies. A previous study has identified the crucial role of residue S296 in hGlyR-α1 potentiation by Δ9-tetrahydrocannabinol (THC) and in cannabinoid-induced analgesia. The recently determined NMR structure of the hGlyR-α1 transmembrane domain (TMD) provides a basis for structure-based virtual screening to discover new analgesic drugs. Using a large ensemble of hGlyR-α1 structures generated from molecular dynamics simulations based on the NMR structure, we screened 1549 FDA approved compounds from the DrugBank database targeted to a cannabinoid binding site near residue S296. Drugs were ranked based on their predicted binding affinities across the ensemble of hGlyR-α1 TMD structures. Four leading compounds were selected for experimental validation in Xenopus laevis oocytes expressing hGlyR-α1. At a low concentration (1 μM), all four leading compounds potentiate hGlyR-α1 currents more than two times, which was greater than that of THC. The hit rate is remarkable. The study provides strong evidence that these leading compounds will be at least as effective as THC for analgesia by acting on hGlyR-α1, but without psychoactive effects. The protocol developed here can easily be applied to the discovery of novel analgesic compounds of even higher efficacy on hGlyRs. Research supported by grants from the NIH and XSEDE.

Membranome: A Database of Single-Spanning Transmembrane Proteins

The Membranome database (http://membranome.org) has been created. The database is focused on structural models of single-spanning (bitopic) transmembrane (TM) proteins and their oligomeric complexes. Current version of Membranome collects data on bitopic proteins of six model organisms, Methanococcus jannaschii, Escherichia coli, Saccharomyces cerevisiae, Dictyostelium discoideum, Arabidopsis thaliana and Homo sapiens that represent different kingdoms of life. It provides comprehensive information on more than 4,500 bitopic proteins, including their three-dimensional (3D) structures, interactions, biological functions, topology, localizations, and classification. The structural data comprise available experimental structures and 3D models of TM alpha-helices that were generated using our new thermodynamic model of alpha-helix formation in the lipid bilayer approximated as an anisotropic solvent with predefined polarity profiles [1]. All bitopic proteins in the Membranome were classified into 15 functional classes, 660 superfamilies and 1076 families. Interactions between different bitopic proteins are included based on data from ConsensusPathDB and PDB. The database provides linking to amino acid sequences from Uniprot and HGNC, structural domains from Interpro and Pfam, protein-proteins interactions from STRING, experimental structures of water-soluble and transmembrane domains from PDB and OPM databases, and annotations in iHOP. Protein entries are also linked to corresponding biological pathways in KEGG, BioCyc and Reactome databases and small molecule metabolic pathways (from SMPDB). Membranome is intended to provide experimentally validated computational models of homo-dimers formed by TM alpha-helices of bitopic proteins.Reference:[1] I.D. Pogozheva, S. Tristram-Nagle, H.I. Mosberg, A.L. Lomize, Structural adaptations of proteins to different biological membranes, Biochim Biophys Acta, 1828 (2013) 2592-2608.