Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer.

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.

Structural modeling of cytokine-receptor-JAK2 signaling complexes using AlphaFold Multimer.

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). They are cell-surface single-pass transmembrane (TM) glycoproteins that regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to TM α-helices of the active TPOR dimer was proposed. The models also help elucidating the molecular basis of oncogenic mutations that may involve non-canonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.

Effects of calreticulin mutations on cell transformation and immunity.

Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK-STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK-STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

Membranome 3.0: Database of single-pass membrane proteins with AlphaFold models.

The Membranome database provides comprehensive structural information on single-pass (i.e., bitopic) membrane proteins from six evolutionarily distant organisms, including protein-protein interactions, complexes, mutations, experimental structures, and models of transmembrane α-helical dimers. We present a new version of this database, Membranome 3.0, which was significantly updated by revising the set of 5,758 bitopic proteins and incorporating models generated by AlphaFold 2 in the database. The AlphaFold models were parsed into structural domains located at the different membrane sides, modified to exclude low-confidence unstructured terminal regions and signal sequences, validated through comparison with available experimental structures, and positioned with respect to membrane boundaries. Membranome 3.0 was re-developed to facilitate visualization and comparative analysis of multiple 3D structures of proteins that belong to a specified family, complex, biological pathway, or membrane type. New tools for advanced search and analysis of proteins, their interactions, complexes, and mutations were included. The database is freely accessible at https://membranome.org.

Comparative Molecular Dynamics Simulation Studies of Realistic Eukaryotic, Prokaryotic, and Archaeal Membranes.

We present a comparative all-atom molecular dynamics simulation study of 18 biomembrane systems with lipid compositions corresponding to eukaryotic, bacterial, and archaebacterial membranes together with three single-component lipid bilayers. A total of 105 lipid types used in this study include diverse sterols and glycerol-based lipids with acyl chains of various lengths, unsaturation degrees, and branched or cyclic moieties. Our comparative analysis provides deeper insight into the influences of sterols and lipid unsaturation on the structural and mechanical properties of these biomembranes, including water permeation into the membrane hydrocarbon core. For sterol-containing membranes, sterol fraction is correlated with the membrane thickness, the area compressibility modulus, and lipid order but anticorrelated with the area per lipid and sterol tilt angles. Similarly, for all 18 biomembranes, lipid order is correlated with the membrane thickness and area compressibility modulus. Sterols and lipid unsaturation produce opposite effects on membrane thickness, but only sterols influence water permeation into the membrane. All membrane systems are accessible for public use in CHARMM-GUI Archive. They can be used as templates to expedite future modeling of realistic cell membranes with transmembrane and peripheral membrane proteins to study their structure, dynamics, molecular interactions, and function in a nativelike membrane environment.

Spatial arrangement of proteins in planar and curved membranes by PPM 3.0.

Cellular protrusions, invaginations, and many intracellular organelles have strongly curved membrane regions. Transmembrane and peripheral membrane proteins that induce, sense, or stabilize such regions cannot be properly fitted into a single flat bilayer. To treat such proteins, we developed a new method and a web tool, PPM 3.0, for positioning proteins in curved or planar, single or multiple membranes. This method determines the energetically optimal spatial position, the hydrophobic thickness, and the radius of intrinsic curvature of a membrane-deforming protein structure by arranging it in a single or several sphere-shaped or planar membrane sections. In addition, it can define the lipid-embedded regions of a protein that simultaneously spans several membranes or determine the optimal position of a peptide in a spherical micelle. The PPM 3.0 web server operates with 17 types of biological membranes and 4 types of artificial bilayers. It is publicly available at https://opm.phar.umich.edu/ppm_server3. PPM 3.0 was applied to identify and characterize arrangements in membranes of 128 proteins with a significant intrinsic curvature, such as BAR domains, annexins, Piezo, and MscS mechanosensitive channels, cation-chloride cotransporters, as well as mitochondrial ATP synthases, calcium uniporters, and TOM complexes. These proteins form large complexes that are mainly localized in mitochondria, plasma membranes, and endosomes. Structures of bacterial drug efflux pumps, AcrAB-TolC, MexAB-OrpM, and MacAB-TolC, were positioned in both membranes of the bacterial cell envelop, while structures of multimeric gap-junction channels were arranged in two opposed cellular membranes.

Thermodynamics-Based Molecular Modeling of α-Helices in Membranes and Micelles.

The Folding of Membrane-Associated Peptides (FMAP) method was developed for modeling α-helix formation by linear peptides in micelles and lipid bilayers. FMAP 2.0 identifies locations of α-helices in the amino acid sequence, generates their three-dimensional models in planar bilayers or spherical micelles, and estimates their thermodynamic stabilities and tilt angles, depending on temperature and pH. The method was tested for 723 peptides (926 data points) experimentally studied in different environments and for 170 single-pass transmembrane (TM) proteins with available crystal structures. FMAP 2.0 detected more than 95% of experimentally observed α-helices with an average error in helix end determination of around 2, 3, 4, and 5 residues per helix for peptides in water, micelles, bilayers, and TM proteins, respectively. Helical and nonhelical residue states were predicted with an accuracy from 0.86 to 0.96, and the Matthews correlation coefficient was from 0.64 to 0.88 depending on the environment. Experimental micelle- and membrane-binding energies and tilt angles of peptides were reproduced with a root-mean-square deviation of around 2 kcal/mol and 7°, respectively. The TM and non-TM states of hydrophobic and pH-triggered α-helical peptides in various lipid bilayers were reproduced in more than 95% of cases. The FMAP 2.0 web server (https://membranome.org/fmap) is publicly available to explore the structural polymorphism of antimicrobial, cell-penetrating, fusion, and other membrane-binding peptides, which is important for understanding the mechanisms of their biological activities.

Mechanism of mutant calreticulin-mediated activation of the thrombopoietin receptor in cancers.

Myeloproliferative neoplasms (MPNs) are frequently driven by mutations within the C-terminal domain (C-domain) of calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Oncogenic transformation requires both mutated CRT and the thrombopoietin receptor (Mpl), but the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. We show that the acquired C-domain of CRTDel52 mediates both Mpl binding and disulfide-linked CRTDel52 dimerization. Cysteine mutations within the novel C-domain (C400A and C404A) and the conserved N-terminal domain (N-domain; C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of CRT oligomers, we identify an N-domain dimerization interface relevant to both WT CRT and CRTDel52. Elimination of disulfide bonds and ionic interactions at both N-domain and C-domain dimerization interfaces is required to abrogate the ability of CRTDel52 to mediate cell proliferation via Mpl. Thus, MPNs exploit a natural dimerization interface of CRT combined with C-domain gain of function to achieve cell transformation.

Aromatic-Amine Pendants Produce Highly Potent and Efficacious Mixed Efficacy µ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Peptidomimetics with Enhanced Metabolic Stability.

We previously reported a novel SAR campaign that converted a metabolically unstable series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist bicyclic core peptidomimetics with promising analgesic activity and reduced abuse liabilities into a more stable series of benzylic core analogues. Herein, we expanded the SAR of that campaign and determined that the incorporation of amines into the benzylic pendant produces enhanced MOR-efficacy in this series, whereas the reincorporation of an aromatic ring into the pendant enhanced MOR-potency. Two compounds, which contain a piperidine (14) or an isoindoline (17) pendant, retained the desired opioid profile in vitro, possessed metabolic half-lives of greater than 1 h in mouse liver microsomes (MLMs), and were active antinociceptive agents in the acetic acid stretch assay (AASA) at subcutaneous doses of 1 mg/kg.

Incorporation of Agouti-Related Protein (AgRP) Human Single Nucleotide Polymorphisms (SNPs) in the AgRP-Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro] Decreases Melanocortin-4 Receptor Antagonist Potency and Results in the Discovery of Melanocortin-5 Receptor Antagonists.

While the melanocortin receptors (MCRs) are known to be involved in numerous biological pathways, the potential roles of the MC5R have not been clearly elucidated in humans. Agouti-related protein (AgRP), an MC3R/MC4R antagonist and MC4R inverse agonist, contains an exposed ß-hairpin loop composed of six residues (Arg-Phe-Phe-Asn-Ala-Phe) that is imperative for binding and function. Within this active loop of AgRP, four human missense polymorphisms were deposited into the NIH Variation Viewer database. These polymorphisms, Arg111Cys, Arg111His, Phe112Tyr, and Ala115Val (AgRP full-length numbering), were incorporated into the peptide macrocycles c[Pro1-Arg2-Phe3-Phe4-Xaa5-Ala6-Phe7-dPro8], where Xaa was Dap5 or Asn5, to explore the functional effects of these naturally occurring substitutions in a simplified AgRP scaffold. All peptides lowered potency at least 10-fold in a cAMP accumulation assay compared to the parent sequences at the MC4Rs. Compounds MDE 6-82-3c, ZMK 2-82, MDE 6-82-1c, ZMK 2-85, and ZMK 2-112 are also the first AgRP-based chemotypes that antagonize the MC5R.

Phospholipid Component Defines Pharmacokinetic and Pharmacodynamic Properties of Synthetic High-Density Lipoproteins.

Synthetic high-density lipoprotein (sHDL) nanoparticles composed of apolipoprotein A-I mimetic peptide and phospholipids have been shown to reduce atherosclerosis in animal models. Cholesterol is mobilized from atheroma macrophages by sHDL into the blood compartment and delivered to the liver for elimination. Historically, sHDL drug discovery efforts were focused on optimizing peptide sequences for interaction with cholesterol cellular transporters rather than understanding how both sHDL components, peptide and lipid, influence its pharmacokinetic and pharmacodynamic profiles. We designed two sets of sHDL having either identical phospholipid but variable peptide sequences with different plasma stability or identical peptide and phospholipids with variable fatty acid chain length and saturation. We found that sHDL prepared with proteolytically stable 22A-P peptide had 2-fold longer circulation half-time relative to the less stable 22A peptide. Yet, longer half-life did not translate into any improvement in cholesterol mobilization. In contrast, sHDL with variable phospholipid compositions showed significant differences in phospholipid PK, with distearoyl phosphatidylcholine-based sHDL demonstrating the longest half-life of 6.0 hours relative to 1.0 hour for palmitoyl-oleoyl phosphatidylcholine-based sHDL. This increase in half-life corresponded to an approx. 6.5-fold increase in the area under the curve for the mobilized cholesterol. Therefore, the phospholipid component in sHDL plays a major role in cholesterol mobilization in vivo and should not be overlooked in the design of future sHDL. SIGNIFICANCE STATEMENT: The phospholipid composition in sHDL plays a critical role in determining half-life and cholesterol mobilization in vivo.

TMPfold: A Web Tool for Predicting Stability of Transmembrane α-Helix Association.

Estimating energies of transmembrane (TM) α-helix association is essential for understanding folding of membrane proteins and formation of their functional assemblies. A new physics-based method was developed and implemented in the TMPfold web server for the calculation of the free energy of TM helix association (ΔGasc) in TM α-bundles of known structure. The method was verified using the experimental ΔGasc values for 36 TM complexes, including dimers of 10 glycophorin A mutants. The calculated free energy changes (ΔΔGasc) caused by mutations in TM helices correlated with experimental changes in the stability of 42 mutants of bacteriorhodopsin and 25 mutants of rhomboid protease. TMPfold was applied for evaluation of ΔGasc in 554 PDB structures of 85 seven-helical TM proteins and identification of stable two-helical folding intermediates. The proposed tentative paths of cotranslational helix assembly of several polytopic proteins were consistent with experimental studies of their folding. TMPfold is accessible at (https://opm.phar.umich.edu/tmpfold_server).

PerMM: A Web Tool and Database for Analysis of Passive Membrane Permeability and Translocation Pathways of Bioactive Molecules.

The PerMM web server and database were developed for quantitative analysis and visualization of passive translocation of bioactive molecules across lipid membranes. The server is the first physics-based web tool that calculates membrane binding energies and permeability coefficients of diverse molecules through artificial and natural membranes (phospholipid bilayers, PAMPA-DS, blood-brain barrier, and Caco-2/MDCK cell membranes). It also visualizes the transmembrane translocation pathway as a sequence of translational and rotational positions of a permeant as it moves across the lipid bilayer, along with the corresponding changes in solvation energy. The server can be applied for prediction of permeability coefficients of compounds with diverse chemical scaffolds to facilitate selection and optimization of potential drug leads. The complementary PerMM database allows comparison of computationally and experimentally determined permeability coefficients for more than 500 compounds in different membrane systems. The website and database are freely accessible at https://permm.phar.umich.edu/ .

Physics-Based Method for Modeling Passive Membrane Permeability and Translocation Pathways of Bioactive Molecules.

Assessment of permeability is a critical step in the drug development process for selection of drug candidates with favorable ADME properties. We have developed a novel physics-based method for fast computational modeling of passive permeation of diverse classes of molecules across lipid membranes. The method is based on heterogeneous solubility-diffusion theory and operates with all-atom 3D structures of solutes and the anisotropic solvent model of the lipid bilayer characterized by transbilayer profiles of dielectric and hydrogen bonding capacity parameters. The optimal translocation pathway of a solute is determined by moving an ensemble of representative conformations of the molecule through the dioleoyl-phosphatidylcholine (DOPC) bilayer and optimizing their rotational orientations in every point of the transmembrane trajectory. The method calculates (1) the membrane-bound state of the solute molecule; (2) free energy profile of the solute along the permeation pathway; and (3) the permeability coefficient obtained by integration over the transbilayer energy profile and assuming a constant size-dependent diffusivity along the membrane normal. The accuracy of the predictions was evaluated against experimental permeability coefficients measured in pure lipid membranes (for 78 compounds, R2 was 0.88 and rmse was 1.15 log units), PAMPA-DS (for 280 compounds, R2 was 0.75 and rmse was 1.59 log units), BBB (for 182 compounds, R2 was 0.69 and rmse was 0.87 log units), and Caco-2/MDCK assays (for 165 compounds, R2 was 0.52 and rmse was 0.89 log units).

Novel Dimethyltyrosine-Tetrahydroisoquinoline Peptidomimetics with Aromatic Tetrahydroisoquinoline Substitutions Show in Vitro Kappa and Mu Opioid Receptor Agonism.

The dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold was originally developed in the production of selective delta opioid receptor (DOR) antagonists. Installation of a 7-benzyl pendant on the tetrahydroisoquinoline core of this classic opioid scaffold introduced kappa opioid receptor (KOR) agonism. Further modification of this pendant resulted in retention of KOR agonism and the addition of mu opioid receptor (MOR) partial agonism, a bifunctional profile with potential to be used in the treatment of cocaine addiction.

Effect of Site-Specific Intermolecular Lysine-Tryptophan Interactions on the Aggregation of Gramicidin-Based Peptides Leading to Pore Formation in Lipid Membranes.

In contrast to the parent pentadecapeptide gramicidin A (gA), some of its cationic analogs have been shown previously to form large-diameter pores in lipid membranes. These pores are permeable to fluorescent dyes, which allows one to monitor pore formation by using the fluorescence de-quenching assay. According to the previously proposed model, the gA analog with lysine substituted for alanine at position 3, [Lys3]gA, forms pores by a homopentameric assembly of gramicidin double-stranded ß-helical dimers. Here, we studied the newly synthesized analogs of [Lys3]gA with single, double and triple substitutions of isoleucines for tryptophans at positions 9, 11, 13, and 15. Replacement of any of the tryptophans of [Lys3]gA with isoleucine resulted in suppression of the pore-forming activity of the peptide, the effect being significantly dependent on the position of tryptophans. In particular, the peptide with a single substitution of tryptophan 13 showed much lower activity than the analogs with single substitutions at positions 9, 11, or 15. Of the peptides with double substitutions, the strongest suppression of the leakage was observed with tryptophans 13 and 15. In the case of triple substitutions, only the peptide retaining tryptophan 11 exhibited noticeable activity. It is concluded that tryptophans 11 and 13 contribute most to pore stabilization in the membrane, whereas tryptophan 9 is not so important for pore formation. Cation-π interactions between the lysine and tryptophan residues of the peptide are suggested to be crucial for the formation of the [Lys3]gA pore.

Membranome 2.0: database for proteome-wide profiling of bitopic proteins and their dimers.

Motivation: Structural studies of TM domains of single-spanning (bitopic) membrane proteins are impeded by their instability, flexibility and heterogeneity. The new computational method TMDOCK allows reliable modeling of homodimers of transmembrane (TM) α-helices on a proteomic scale. Results: 3D models of 2129 parallel homodimers formed by TM α-helices of bitopic proteins from six evolutionarily distant organisms were modeled by TMDOCK, verified through experimental data available for nearly 600 proteins, and included in the Membranome database (v.2.0) along with related information to facilitate structural and evolutionary analysis of bitopic proteins. Availability and implementation: http://membranome.org. Contact: almz@umich.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Evolution and adaptation of single-pass transmembrane proteins.

A comparative analysis of 6039 single-pass (bitopic) membrane proteins from six evolutionarily distant organisms was performed based on data from the Membranome database. The observed repertoire of bitopic proteins is significantly enlarged in eukaryotic cells and especially in multicellular organisms due to the diversification of enzymes, emergence of proteins involved in vesicular trafficking, and expansion of receptors, structural, and adhesion proteins. The majority of bitopic proteins in multicellular organisms are located in the plasma membrane (PM) and involved in cell communication. Bitopic proteins from different membranes significantly diverge in terms of their biological functions, size, topology, domain architecture, physical properties of transmembrane (TM) helices and propensity to form homodimers. Most proteins from eukaryotic PM and endoplasmic reticulum (ER) have the N-out topology. The predicted lengths of TM helices and hydrophobic thicknesses, stabilities and hydrophobicities of TM α-helices are the highest for proteins from eukaryotic PM, intermediate for proteins from prokaryotic cells, ER and Golgi apparatus, and lowest for proteins from mitochondria, chloroplasts, and peroxisomes. Tyr and Phe residues accumulate at the cytoplasmic leaflet of PM and at the outer leaflet of membranes of bacteria, Golgi apparatus, and nucleus. The propensity for dimerization increases from unicellular to multicellular eukaryotes, from enzymes to receptors, and from intracellular membrane proteins to PM proteins. More than half of PM proteins form homodimers with a 2:1 ratio of right-handed to left-handed helix packing arrangements. The inverse ratio (1:2) was observed for dimers from the ER, Golgi and vesicles.

Placement of Hydroxy Moiety on Pendant of Peptidomimetic Scaffold Modulates Mu and Kappa Opioid Receptor Efficacy.

In an effort to expand the structure-activity relationship (SAR) studies of a series of mixed-efficacy opioid ligands, peptidomimetics that incorporate methoxy and hydroxy groups around a benzyl or 2-methylindanyl pendant on a tetrahydroquinoline (THQ) core of the peptidomimetics were evaluated. Compounds containing a methoxy or hydroxy moiety in the o- or m-positions increased binding affinity to the kappa opioid receptor (KOR), whereas compounds containing methoxy or hydroxy groups in the p-position decreased KOR affinity and reduced or eliminated efficacy at the mu opioid receptor (MOR). The results from a substituted 2-methylindanyl series aligned with the findings from the substituted benzyl series. Our studies culminated in the development of 8c, a mixed-efficacy MOR agonist/KOR agonist with subnanomolar binding affinity for both MOR and KOR.

TMDOCK: An Energy-Based Method for Modeling α-Helical Dimers in Membranes.

TMDOCK is a novel computational method for the modeling of parallel homodimers formed by transmembrane (TM) α-helices. Three-dimensional (3D) models of dimers are generated by threading a target amino acid sequence through several structural templates, followed by local energy minimization. This is the first method that identifies helix dimerization modes and ranks them based on the calculated free energy of α-helix association. Free energy components include van der Waals, hydrogen bonding, and dipole interactions; side-chain conformational entropy; and solvation energy in the anisotropic lipid environment. TMDOCK reproduced 26 experimental dimeric structures formed by TM α-helices of 21 single-pass membrane proteins (including 4 mutants) with Cα atom rmsd from 1.0 to 3.3Å. Assessment of dimerization heterogeneity of these TM domains demonstrated that 7 of them have a unique dimer structure, 12 have at least 2 alternative conformations, and 2 have a large number of different association modes. All unique experimental structures of proteins from the first group and eight structures from the second group were reproduced in computations as top-ranked models. A fast version of the method is available through the web server (http://membranome.org/tm_server.php).