Predicting the Assembly of the Transmembrane Domains of Viral Channel Forming Proteins and Peptide Drug Screening Using a Docking Approach.

A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.

Rotational Dynamics of The Transmembrane Domains Play an Important Role in Peptide Dynamics of Viral Fusion and Ion Channel Forming Proteins-A Molecular Dynamics Simulation Study.

Focusing on the transmembrane domains (TMDs) of viral fusion and channel-forming proteins (VCPs), experimentally available and newly generated peptides in an ideal conformation of the S and E proteins of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and SARS-CoV, gp41 and Vpu, both of human immunodeficiency virus type 1 (HIV-1), haemagglutinin and M2 of influenza A, as well as gB of herpes simplex virus (HSV), are embedded in a fully hydrated lipid bilayer and used in multi-nanosecond molecular dynamics simulations. It is aimed to identify differences in the dynamics of the individual TMDs of the two types of viral membrane proteins. The assumption is made that the dynamics of the individual TMDs are decoupled from their extra-membrane domains, and that the mechanics of the TMDs are distinct from each other due to the different mechanism of function of the two types of proteins. The diffusivity coefficient (DC) of the translational and rotational diffusion is decreased in the oligomeric state of the TMDs compared to those values when calculated from simulations in their monomeric state. When comparing the calculations for two different lengths of the TMD, a longer full peptide and a shorter purely TMD stretch, (i) the difference of the calculated DCs begins to level out when the difference exceeds approximately 15 amino acids per peptide chain, and (ii) the channel protein rotational DC is the most affected diffusion parameter. The rotational dynamics of the individual amino acids within the middle section of the TMDs of the fusion peptides remain high upon oligomerization, but decrease for the channel peptides, with an increasing number of monomers forming the oligomeric state, suggesting an entropic penalty on oligomerization for the latter.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy.

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Membrane partitioning of peptide aggregates: coarse-grained molecular dynamics simulations.

Coarse-grained molecular dynamics (CGMD) simulation technique (MARTINI force field) is applied to monitor the aggregation of helical peptides representing the transmembrane sequence and its extension of bone marrow stromal cell antigen 2 (BST-2). One of the peptides is coupled with a protein transducing domain (PTD) of nine arginine residues (R9) at its N-terminal side as well as a peptide, pep11**, which has been shown to bind to human papilloma virus 16 (HPV16) E6 oncoprotein. A short hydrophobic stretch of the transmembrane domain (TMD) of BST-2 aggregates the fastest and inserts into a lipid membrane. An aggregate of R9-pep11** attaches to the membrane via simultaneous contact of many arginine residues. Monomers from the aggregates of the shortest of the hydrophobic TMDs dissolve into the opposing leaflet when the aggregate spans the bilayer. A 'flipping' of the individual monomeric peptides is not observed.Communicated by Ramaswamy H. Sarma.

Interaction of antivirals with a heptameric bundle model of the p7 protein of hepatitis C virus.

A series of ligands are known experimentally to affect the infectivity cycle of the hepatitis C virus. The target protein for the ligands is proposed to be p7, a 63 amino acid polytopic channel-forming protein, with possibly two transmembrane domains. Protein p7 is found to assemble into functional oligomers of various sizes, depending on the genotype (GT). Nine ligands are docked to various sites of a computationally derived heptameric bundle of p7 of GT1a. The energy of interaction, here binding energy, is calculated using three different docking programs (Autodock, MOE, LeadIT). Three protein regions are defined to which the ligands are placed, the loop region and the site with the termini as well as the mid-region which is supposed to track poses inside the putative pore. A common feature is that the loop sites and poses either within the pore or at the intermonomer space of the bundle are preferred for all ligands with proposed binding energies smaller than -10 kJ/mol. BIT225, benzamine, amantadine, and NN-DNJ show good overall scoring.

Specification of binding modes between a transmembrane peptide mimic of ATP6V0C and polytopic E5 of human papillomavirus-16.

Interaction of E5 of papillomavirus-16 based on its three transmembrane domains (TMDs) with a peptide mimicking the fourth TMD (TMD-A) of the 16 kDa c subunit of the human vacuolar H+-ATPase, ATP6V0C, and one of its mutant is investigated. Docking reveals binding of the peptide between the second and third TMD of E5. A series of hydrophobic residues are responsible for the contact. Estimated weak binding energies based on potential of mean force calculations reveal marginal differences of the estimated binding energies between wild type (WT) and mutant peptide. Also differences in estimated binding energies of dimers of the individual TMDs of E5 with the WT peptide are marginal. Correlation of rotational data derived from coarse-grained molecular dynamics simulations of the peptides and the protein as well as from the principal component analysis reveal that the binding of TMD-A with TMD3 is enthalpy driven and binding with TMD2 is guided by entropic conditions.

Decoupled side chain and backbone dynamics for proton translocation - M2 of influenza A.

The 97 amino acid bitopic membrane protein M2 of influenza A forms a tetrameric bundle in which two of the monomers are covalently linked via a cysteine bridge. In its tetrameric assembly the protein conducts protons across the viral envelope and within intracellular compartments during the infectivity cycle of the virus. A key residue in the translocation of the protons is His-37 which forms a planar tetrad in the configuration of the bundle accepting and translocating the incoming protons from the N terminal side, exterior of the virus, to the C terminal side, inside the virus. With experimentally available data from NMR spectroscopy of the transmembrane domains of the tetrameric M2 bundle classical MD simulations are conducted with the protein bundle in different protonation stages in respect to His-37. A full correlation analysis (FCA) of the data sets with the His-37 tetrad either in a fully four times unprotonated or protonated state, assumed to mimic high and low pH in vivo, respectively, in both cases reveal asymmetric backbone dynamics. His-37 side chain rotation dynamics is increased at full protonation of the tetrad compared to the dynamics in the fully unprotonated state. The data suggest that proton translocation can be achieved by decoupled side chain or backbone dynamics. Graphical abstract Visualization of the tetrameric bundle of the transmembrane domains of M2 of influenza A after 200 ns of MD simulations (upper left). The four histidine residues 37 are either not protonated as in M20 or fully protonated is in M24+. The asymmetric dynamics of the backbones are shown after a full correlation analysis (FCA) in blue (lower left). The rotational dynamics of the χ2 dihedral angles of the histidines in M20 (upper right) are less than those in M24+ (lower right).

Lipid raft-associated stomatin enhances cell fusion.

Membrane fusions that occur during vesicle transport, virus infection, and tissue development, involve receptors that mediate membrane contact and initiate fusion and effectors that execute membrane reorganization and fusion pore formation. Some of these fusogenic receptors/effectors are preferentially recruited to lipid raft membrane microdomains. Therefore, major constituents of lipid rafts, such as stomatin, may be involved in the regulation of cell-cell fusion. Stomatin produced in cells can be released to the extracellular environment, either through protein refolding to pass across lipid bilayer or through exosome trafficking. We report that cells expressing more stomatin or exposed to exogenous stomatin are more prone to undergoing cell fusion. During osteoclastogenesis, depletion of stomatin inhibited cell fusion but had little effect on tartrate-resistant acid phosphatase production. Moreover, in stomatin transgenic mice, increased cell fusion leading to enhanced bone resorption and subsequent osteoporosis were observed. With its unique molecular topology, stomatin forms molecular assembly within lipid rafts or on the appositional plasma membranes, and promotes membrane fusion by modulating fusogenic protein engagement.-Lee, J.-H., Hsieh, C.-F., Liu, H.-W., Chen, C.-Y., Wu, S.-C., Chen, T.-W., Hsu, C.-S., Liao, Y.-H., Yang, C.-Y., Shyu, J.-F., Fischer, W. B., Lin, C.-H. Lipid raft-associated stomatin enhances cell fusion.

Weak Selectivity Predicted for Modeled Bundles of Viral Channel-Forming Protein E5 of Human Papillomavirus-16.

Protein E5 is a polytopic 83 amino acid membrane protein with three transmembrane domains (TMDs), encoded by high-risk human papillomavirus-16 (HPV-16). HPV-16 is found to be the causative agent for cervical cancer. Protein E5, among other proteins (e.g., E6, E7), is expressed at an "early" (E) stage when the cell turns malignant. It has been experimentally found that E5 forms hexameric assemblies, which show the characteristics of the class of so-called channel-forming proteins by rendering lipid membranes permeable to ions and small molecules. Protein E5 is used to achieve structural models of the protein in assembled bundles using a force field-based docking approach. Extended molecular dynamics simulations of selected bundles in fully hydrated lipid bilayers suggest the second TMD to be pore-lining, allowing for water columns to exist within the lumen of the pore. Full correlation analysis indicates asymmetric dynamics within the monomers of the bundle. Potential of mean force calculations of a snapshot structure of the putative open pore of the protein bundle propose low selectivity.

Membrane protein assembly: two cytoplasmic phosphorylated serine sites of Vpu from HIV-1 affect oligomerization.

Viral protein U (Vpu) encoded by human immunodeficiency virus type 1 (HIV-1) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the HIV-1 infectivity cycle. In this study, full-length Vpu (M group) from clone NL4-3 was over-expressed in human cells and purified in an oligomeric state. Various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. Size exclusion chromatography of wild-type Vpu and mutants indicated that the smallest assembly unit of Vpu was a dimer and over time Vpu formed higher oligomers. The rate of oligomerization increased when (i) the degree of phosphorylation at serines 52 and 56 was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of Vpu affects oligomerization. Coarse-grained molecular dynamic simulations with models of wild-type and mutant Vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of Vpu also modulate oligomerization.

Small molecule ligand docking to genotype specific bundle structures of hepatitis C virus (HCV) p7 protein.

The genome of hepatitis C virus encodes for an essential 63 amino acid polytopic protein p7 of most likely two transmembrane domains (TMDs). The protein is identified to self-assemble thereby rendering lipid membranes permeable to ions. A series of small molecules such as adamantanes, imino sugars and guanidinium compounds are known to interact with p7. A set of 9 of these small molecules is docked against hexameric bundles of genotypes 5a (bundle-5a) and 1b (bundle-1b) using LeadIT. Putative sites for bundle-5a are identified within the pore and at pockets on the outside of the bundle. For bundle-1b preferred sites are found at the site of the loops. Binding energies are in favour of the guanidinium compounds. Rescoring of the identified poses with HYDE reveals a dehydration penalty for the guanidinium compounds, leaving the adamantanes and imino sugar in a better position. Binding energies calculated by HYDE and those by LeadIT indicate that all compounds are moderate binders.

Asymmetric dynamics of ion channel forming proteins - Hepatitis C virus (HCV) p7 bundles.

Protein p7 of hepatitis C virus (HCV) is a short 63 amino acid membrane protein which homo-oligomerises in the lipid membrane to form ion and proton conducting bundles. Two different genotypes (GTs) of p7, 1a and 5a, are used to simulate hexameric bundles of the protein embedded in a fully hydrated lipid bilayer during 400 ns molecular dynamics (MD) simulations. Whilst the bundle of GT 1a is based on a fully computational derived structure, the bundle of GT 5a is based on NMR spectroscopic data. Results of a full correlation analysis (FCA) reveal that albeit structural differences both bundles screen local minima during the simulation. The collective motion of the protein domains is asymmetric. No 'breathing-mode'-like dynamics is observed. The presence of divalent ions, such as Ca-ions affects the dynamics of especially solvent exposed parts of the protein, but leaves the asymmetric domain motion unaffected.

Patch formation of a viral channel forming protein within a lipid membrane--Vpu of HIV-1.

Ion channels and their viral companions are defined by their quaternary structure. The individual sub-units have to assemble into homo- or hetero-oligomers. Using Vpu of HIV-1, a putative viral channel forming protein (VCP), as a test case, the formation of a quaternary structure is monitored using coarse grained molecular dynamics (CGMD) simulations. Full length Vpu is generated by combining the helical transmembrane domain (TMD) with the cytoplasmic domain derived from NMR spectroscopy. Patches of 2 to 6 as well as patches of 16 and 32 Vpu proteins, Vpu-WT, containing unphosphorylated serines 52 and 56 are used to study assembly dynamics. The same patches are simulated for the Vpu double mutant, Vpu-DD, in which the two serines 52 and 56 are replaced by aspartic acid. Serines 52 and 56 in Vpu-WT allow short lived contacts between the cytoplasmic domains. Dimer formation is the first step for long lasting assemblies and is induced by the EYR motif. Roll-over movements allow rearrangement within the dimer. Independent of the number of Vpu proteins, Vpu-DD prefers smaller aggregates than Vpu-WT. In the case of simulation of 4 Vpu-WT proteins a pore-like assembly is directly identified with the TMD Ser-23 pointing towards a putative central pore axis.

Interaction of ions with the luminal sides of wild-type and mutated skeletal muscle ryanodine receptors.

Ryanodine receptors (RyRs) are the largest known ion channels, and are of central importance for the release of Ca(2+) from the sarco/endoplasmic reticulum (SR/ER) in a variety of cells. In cardiac and skeletal muscle cells, contraction is triggered by the release of Ca(2+) into the cytoplasm and thus depends crucially on correct RyR function. In this work, in silico mutants of the RyR pore were generated and MD simulations were conducted to examine the impact of the mutations on the Ca(2+) distribution. The Ca(2+) distribution pattern on the luminal side of the RyR was most affected by G4898R, D4899Q, E4900Q, R4913E, and D4917A mutations. MD simulations with our wild-type model and various ion species showed a preference for Ca(2+) over other cations at the luminal pore entrance. This Ca(2+)-accumulating characteristic of the luminal RyR side may be essential to the conductance properties of the channel.

Viral channel forming proteins--How to assemble and depolarize lipid membranes in silico.

Viral channel forming proteins (VCPs) have been discovered in the late 70s and are found in many viruses to date. Usually they are small and have to assemble to form channels which depolarize the lipid membrane of the host cells. Structural information is just about to emerge for just some of them. Thus, computational methods play a pivotal role in generating plausible structures which can be used in the drug development process. In this review the accumulation of structural data is introduced from a historical perspective. Computational performances and their predictive power are reported guided by biological questions such as the assembly, mechanism of function and drug-protein interaction of VCPs. An outlook of how coarse grained simulations can contribute to yet unexplored issues of these proteins is given. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Estimating binding free energy of a putative growth factors EGF-VEGF complex - a computational bioanalytical study.

Epidermal growth factor (EGF) and homodimeric vascular endothelial growth factor (VEGF) bind to cell surface receptors. They are responsible for cell growth and angiogenesis, respectively. Docking of the individual proteins as monomeric units using ZDOCK 2.3.2 reveals a partial blocking of the receptor binding site of VEGF by EGF. The receptor binding site of EGF is not affected by VEGF. The calculated binding energy is found to be intermediate between the binding energies calculated for Alzheimer's Aß42 and the barnase/barstar complex.

Development and Characterization of the Recombinant Human VEGF-EGF Dual-Targeting Fusion Protein as a Drug Delivery System.

The design, preparation, as well as structural and functional characterizations of the recombinant fusion protein hVEGF-EGF as a dual-functional agent that may target both EGFR (R: receptor) and angiogenesis are reported. hVEGF-EGF was found to bind to EGFR more strongly than did EGF, and to bind to VEGFR similarly to VEGF. Mass spectrometry measurements showed that the sites of DTPA (diethylenetriaminepentaacetic acid) conjugated hVEGF-EGF (for radiolabeling) were the same as those of its parent hEGF and hVEGF proteins. All DTPA-conjugated proteins retained similar binding capacities to their respective receptors as compared to their respective parent proteins. In vitro cell binding studies using BAEC (a bovine aortic endothelial cell) and MDA-MB-231 (a human breast cancer) cells expressing both EGFR and VEGFR confirmed similar results. Treating BAEC cells with hVEGF-EGF induced remarkable phosphorylation of EGFR, VEGFR, and their downstream targets ERK1/2. Nevertheless, the radiolabeled (111)In-DTPA-hVEGF-EGF showed cytotoxicity against MDA-MB-231 cells. Pharmacokinetic studies using (111)In-DTPA-hVEGF-EGF in BALB/c nude mice showed that appreciable tracer activities were accumulated in liver and spleen. In all, this study demonstrated that the fusion protein hVEGF-EGF maintained the biological specificity toward both EGFR and VEGFR and may be a potential candidate as a dual-targeting moiety in developing anticancer drugs.

Genotype-specific differences in structural features of hepatitis C virus (HCV) p7 membrane protein.

The 63 amino acid polytopic membrane protein, p7, encoded by hepatitis C virus (HCV) is involved in the modulation of electrochemical gradients across membranes within infected cells. Structural information relating to p7 from multiple genotypes has been generated in silico (e.g. genotype (GT) 1a), as well as obtained from experiments in form of monomeric and hexameric structures (GTs 1b and 5a, respectively). However, sequence diversity and structural differences mean that comparison of their channel gating behaviour has not thus far been simulated. Here, a molecular model of the monomeric GT 1a protein is optimized and assembled into a hexameric bundle for comparison with both the 5a hexamer structure and another hexameric bundle generated using the GT 1b monomer structure. All bundles tend to turn into a compact structure during molecular dynamics (MD) simulations (Gromos96 (ffG45a3)) in hydrated lipid bilayers, as well as when simulated at 'low pH', which may trigger channel opening according to some functional studies. Both GT 1a and 1b channel models are gated via movement of the parallel aligned helices, yet the scenario for the GT 5a protein is more complex, with a short N-terminal helix being involved. However, all bundles display pulsatile dynamics identified by monitoring water dynamics within the pore.

Structure based computational assessment of channel properties of assembled ORF-8a from SARS-CoV.

ORF 8a is a short 39 amino acid bitopic membrane protein encoded by severe acute respiratory syndrome causing corona virus (SARS-CoV). It has been identified to increase permeability of the lipid membrane for cations. Permeability is suggested to occur due to the assembly of helical bundles. Computational models of a pentameric assembly of 8a peptides are generated using the first 22 amino acids, which include the transmembrane domain. Low energy structures reveal a hydrophilic pore mantled by residues Thr-8, and -18, Ser-11, Cys-13, and Arg-22. Potential of mean force (PMF) profiles for mono (Na(+) , K(+) , Cl(-) ) and divalent (Ca(2+) ) ions along the pore are calculated. The data support experimental findings of a weak cation selectivity of the channel. Calculations on 8a are compared to data derived for a pentameric bundle consisting of the M2 helices of the bacterial pentameric ligand gated ion channel GLIC (3EHZ). PMF curves of both, bundles 8a and M2, show sigmoidal shaped profiles. In comparison to the data for the M2-GLIC model, data of the 8a bundle show lower amplitude of the PMF values between maximum and minimum and less discrimination amongst ions.

Docking assay of small molecule antivirals to p7 of HCV.

Protein p7 of HCV is a 63 amino acid channel forming membrane protein essential for the progression of viral infection. With this momentousness, p7 emerges as an important target for antiviral therapy. A series of small molecule drugs, such as amantadine, rimantadine, amiloride, hexamethylene amiloride, NN-DNJ and BIT225 have been found to affect the channel activity. These compounds are docked against monomeric and hexameric structures of p7 taken at various time steps from a molecular dynamics simulation of the protein embedded in a hydrated lipid bilayer. The energetics of binding identifies the guanidine based ligands as the most potent ligands. The adamantanes and NN-DNJ show weaker binding energies. The lowest energy poses are those at the site of the loop region for the monomer and hexamer. For the latter, the poses show a tendency of the ligand to face the lumen of the pore. The mode of binding is that of a balance between hydrophobic interactions and hydrogen bond formation with backbone atoms of the protein.