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The initial step of interaction of some pathogens with the host is driven by the interaction of glycoproteins of either side via endcaps of their glycans. These end caps consist of sialic acids or sugar molecules. Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are found to use this route of interaction. The strength and spatial interactions on the single molecule level of sialic acids with either the spike (S) protein of SARS coronaviruses, or human angiotensin-converting enzyme 2 (ACE2) and furin are probed and compared to the binding modes of those sugar molecules which are present in glycans of glycoproteins. The protocol of using single molecules is seen as a simplified but effective mimic of the complex mode of interaction of the glycans. Averaged estimated binding energies from a docking approach result in preferential binding of the sialic acids to a specific binding site of the S protein of human coronavirus OC43 (HCoV-OC43). Furin is proposed to provide better binding sites for sialic acids than ACE2, albeit outweighed by sites for other sugar molecules. Absolute minimal estimated binding energies indicate weak binding affinities and are indifferent to the type of sugar molecules and the proteins. Neither the proposed best binding sites of the sialic acids nor those of the sugar molecules overlap with any of the cleavage sites at the S protein and the active sites of the human proteins.

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

Horizontal transfer of mobile genetic elements, such as integrating and conjugative elements (ICEs), plays an important role in generating diversity and maintaining comprehensive pan-genomes in bacterial populations. The human gastric pathogen Helicobacter pylori, which is known for its extreme genetic diversity, possesses highly efficient transformation and recombination systems to achieve this diversity, but it is unclear to what extent these systems influence ICE physiology. In this study, we have examined the excision/integration and horizontal transfer characteristics of an ICE (termed ICEHptfs4) in these bacteria. We show that transfer of ICEHptfs4 DNA during mating between donor and recipient strains is independent of its conjugation genes, and that homologous recombination is much more efficient than site-specific integration into the recipient chromosome. Nevertheless, ICEHptfs4 excision by site-specific recombination occurs permanently in a subpopulation of cells and involves relocation of a circularization-dependent promoter. Selection experiments for excision indicate that the circular form of ICEHptfs4 is not replicative, but readily reintegrates by site-specific recombination. Thus, although ICEHptfs4 harbours all essential transfer genes, and typical ICE functions such as site-specific integration are active in H. pylori, canonical ICE transfer is subordinate to the more efficient general DNA uptake and homologous recombination machineries in these bacteria.

Display Omitted Binding poses of antivirals to hexameric bundles of p7 of HCV genotype 5a and 1b.LeadIT suggests guanidinium compounds then imino sugars, adamantanes.Rescoring with HYDE reverses the order.Favored binding sites within the pore and at outside pockets for bundle-5a.Favored binding sites at the sites of the loops for bundle-1b. 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.

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.

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., Graphical abstract, Highlights • Early references about the discovery of viral channel forming proteins. • Latest structural information about the class of proteins. • Identification of structural motifs, assembly mechanism of function and drug action using computational methods. • Outlook for the use of coarse grained techniques to address assembly and integration into cellular processes.

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.

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.

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.

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., Graphical abstract, Highlights • Genotype specific structural features of hexameric bundles of p7 of HCV • In silico built models of genotype 1a bundle compared with models of genotypes 1b and 5a from NMR • Genotype 1a and 1b channels are gated via movement of the parallel aligned helices. • Complex scenario for genotype 5a bundle model • All bundles show pulsatile dynamics.

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.

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.

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 M2

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.

Viral proteins assemble into homopolymers in the infected cells and have a role as diffusion-amplifier for ions across subcellular membranes. The homopolymer of hepatitis C virus, protein p7 of strain 1a, which is known to form channels, is used to investigate the dynamics of physiological relevant ions, Na(+), K(+), Cl(-) and Ca(2+) in the vicinity of the protein bundle. The protein bundle is generated by a combination of docking approach and molecular dynamics (MD) simulations. Ion dynamics are recorded during multiple 200 ns MD simulations of 1M solutions. His-17 is found to point into the lumen of the pore. Protonation of this residue allows Cl-ions to enter the pore while in its unprotonated state Ca-ions are found within the pore as well. Applied voltage identifies large Cl-ion currents from the site of the loop passing through the pore. Rectification of the current of the Cl-ions is observed.

Vpu is an 81 amino acid type I integral membrane protein encoded by human immunodeficiency virus type 1 (HIV-1). It is identified to support viral release by potentially forming ion and substrate conducting channels and by modulating the function of host factors. The focus is on the interaction of the transmembrane domains of Vpu with those of host factors using a combination of molecular dynamics simulations and docking approach. Binding poses and adopted tilt angles of the dimers are analyzed and correlated with experimentally derived activity data from literature. Vpu activity is driven by dimerization with the host protein via its alanine rim Ala-8/11/15/19. Tight binding is shown by an almost parallel alignment of the helices in the dimers. Less parallel alignment is proposed to correlate with lower activity. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.

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.

Protein 3a is a 274 amino acid polytopic channel protein with three putative transmembrane domains (TMDs) encoded by severe acute respiratory syndrome corona virus (SARS-CoV). Synthetic peptides corresponding to each of its three individual transmembrane domains (TMDs) are reconstituted into artificial lipid bilayers. Only TMD2 and TMD3 induce channel activity. Reconstitution of the peptides as TMD1 1TMD3 as well as TMD2 1TMD3 in a 1 : 1 mixture induces membrane activity for both mixtures. In a 1 : 1 : 1 mixture, channel like behavior is almost restored. Expression of full length 3a and reconstitution into artificial lipid bilayers reveal a weak cation selective (PK � 2P Cl) rectifying channel. In the presence of nonphysiological concentration of Ca-ions the channel develops channel activity. V C 2013 Wiley Periodicals, Inc. Biopolymers 99: 628‐635, 2013.

Different routes of assembly are probed for the transmembrane domain (TMD) of the bitopic membrane protein Vpu from HIV-1. Vpu is responsible for the amplification of viral release from the host cell. The mode of action includes (i) heteroassembly with host factors and (ii) the formation of homo-oligomers, which are able to conduct ions across the lipid membrane. Two different routes of assembling short sequences of the N terminus, including the TMD of Vpu, Vpu1–32, and Vpu8–26, are presented by using a combination of classical molecular dynamics (MD) simulations combined with a docking approach. The rim of alanines (Ala-8, -11, -15, and -19) resembles an interlocking motif for the sequential assembly into a dimer and trimer. Simultaneous assembly results in oligomeric bundles (trimers to pentamers) with either tryptophans (Trp-23) or purely hydrophobic residues facing the center. Bundles, with serines facing the pore (Ser-24), are energetically not the lowest structures. For pentameric bundles with Ser-24 facing the pore, no water column develops during a short 25 ns MD simulation. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 517–529, 2013.

We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.

2B is a 99 amino acid membrane protein encoded by enteroviruses such as polio and coxsackie viruses with two transmembrane domains. The protein is found to make membranes of infected cells permeable. Using a computational approach which positions the models and assesses stability by molecular dynamics (MD) simulations a putative tetrameric bundle model of 2B is generated. The bundles show a pore lining motif of three lysines followed by a serine. The bundle is discussed in terms of different possible orientations of the helices in the membrane and the consequences this has on the in vivo activity of 2B.

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.

Sequence alignment is an important tool for identifying regions of similarities among proteins and for, thus, establishing functional and structural relationships between different proteins. Here, alignments of transmembrane domains (TMDs) of viral channel forming proteins with host ion channels and toxins are evaluated. The following representatives of polytopic viral channel proteins are chosen: (i) p7 of HCV and 2B of Polio virus (two TMDs) and (ii) 3a of SARS-CoV (three TMDs). Using ClustalW2, each of the TMDs of the viral channels is aligned, and the overlap is mapped onto structural models of the host channels and toxins focusing on the pore-lining TMDs. The analysis reveals that p7 and 2B TMDs align with the pore-facing TMD of MscL, and 3a-TMDs align with those of ligand-gated ion channels. Possible implications concerning the mechanism of function of the viral proteins are discussed.

ORF 3a of human severe acute respiratory syndrome corona virus (SARS-CoV) has been identified as a 274 amino acid membrane protein. When expressed in Xenopus oocytes the protein forms channels. Based on bioinformatics approaches the topology has been identified to include three transmembrane domains (TMDs). Since structural models from experiments are still lacking, computational methods can be challenged to generate such models. In this study, a ‘sequential approach’ for the assembly is proposed in which the individual TMDs are assembled one by one. This protocol is compared with a concerted protocol in which all TMDs are assembled simultaneously. The role of the loops between the TMDs during assembly of the monomers into a bundle is investigated. Molecular dynamics simulations for 20 ns are performed as a short equilibration to assess the bundle stability in a lipid environment. The results suggest that bundles are likely with the second TMD facing the putative pore. All the putative bundles show water molecules trapped within the lumen of the pore with only occasional events of complete crossing. Electronic supplementary material The online version of this article (doi:10.1007/s00894-011-1092-6) contains supplementary material, which is available to authorized users.

The cellular and subcellular membranes encounter an important playground for the activity of membrane proteins encoded by viruses. Viral membrane proteins, similar to their host companions, can be integral or attached to the membrane. They are involved in directing the cellular and viral reproduction, the fusion and budding processes. This review focuses especially on those integral viral membrane proteins which form channels or pores, the classification to be so, modeling by in silico methods and potential drug candidates. The sequence of an isolate of Vpu from HIV-1 is aligned with host ion channels and a toxin. The focus is on the alignment of the transmembrane domains. The results of the alignment are mapped onto the 3D structures of the respective channels and toxin. The results of the mapping support the idea of a ‘channel–pore dualism’ for Vpu.

Viren manipulieren den Stoffwechsel einer infizierten Zelle zu ihren Gunsten. Dafur nutzen sie kleine Proteine, die Ionenkanale bilden. Doch wie lagern sich diese Proteine zu funktionellen, membrandurchspannenden Einheiten zusammen? Leitfahigkeitsmessungen und Computersimulation sind zwei der vielen Methoden, die dazu beitragen, die Vorgange zu verstehen und letztlich neue antivirale Wirkstoffe zu entwerfen.

The generation of computational models is an alternative route to obtain reliable structures for the oligomeric state of membrane proteins. A strategy has been developed to search the conformational space of all possible assemblies in a reasonable time, taking symmetry considerations into account. The methodology tested on M2 from influenza A, shows an excellent agreement with established structures. For Vpu from HIV-1 a series of conformational distinct structures are proposed. For the first time a structural model for a fully assembled transmembrane part of 3a from SARS-CoV is proposed.

The dynamic behavior of monomeric Vpu(1-32) from HIV-1 in different lipid environments has been studied. The peptide shows highly flexible behavior during the simulations and easily adapts to changing lipid environments as it experiences when travelling through the Golgi apparatus. Protein-lipid interactions do not show any significant correlation towards lipid type or thickness based on multiple 10 ns simulations. The averaged structure of a series of 16 independent simulations suggest kink around Ser-24, which compensates the polarity of its side chain by forming hydrogen bonds with the carbonyl backbone of adjacent amino acids towards the N-terminus.

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.

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.

VPU of HIV has been verified as a channel-forming protein by experiment. The self-assembly process and the oligomer state of VPU are not clear. We use coarse-graining molecular dynamics simulation to study the full-length channel-forming protein VPU in lipid bilayer. The full-length of VPU was constructed by linking the cytoplasm and transmembrane domain solved by NMR. We put different number of VPUs 2, 3, 4, 5 or bigger amount 16, 32 in simulation box. Our results show several binding sites and the oligomer state. In those simulations, the four VPUs form a symmetric bundle which is close to the channel structure. The tetramer was seem to form via a dimer. Not only transmembrane domain but also cytoplasm domain was involved in self-assembly.

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelial cells leading to precancerous lesions. The HPV genome encodes three oncoproteins: E5, E6 and E7 from which E5 is the least understood. E5 of HPV-16, one of the “high risk” types of HPV strains, is an 83 amino acid hydrophobic membrane protein, with three hydrophobic transmembrane domains (TMDs). It oligomerizes into dimers or higher oligomers which form ion channels most likely by forming hexameric bundles. Computational modeling is used to obtain structural and functional features of this protein.The three TMDs of E5 are identified using secondary structure prediction programs. The TMDs are assembled into a monomer by a ‘Sequential’ and ‘Simultaneous’ docking approach in which the conformational space of the three helices is screened by simultaneously altering distance, tilt and rotational angle between them. In a consequent step, loops linking the three helices are added using the program Loopy. Finally six monomers are assembled into a hexameric bundle. The bundle with TMD2 lining the pore remains intact allowing formation water filled pocket during entire 100 ns MD simulations. The water pocket formed by the six TMD2s of the bundle is mostly mantled by hydrophilic residues such as Ser-35, −37 and Thr-38, −40. Bundles with the other two TMDs, TMD1 and TMD3, mantling the pore are energetically almost undistiguishable from the bundle with TMD2 facing the pore.With TMD2s facing the pore, ion channel activity possible. All asymmetric bundle architectures account for interactions of E5 with host proteins.

Vpu is an 81 amino acid protein of HIV-1 with two phosphorylation sites. It consists of a short N-terminal end traversing the bilayer and a longer cytoplasmic part. The dual functional role of Vpu is attributed to these topological distinct regions of the protein. The first 52 amino acids of Vpu (HV1H2) have been simulated, which are thought to be embedded in a fully hydrated lipid bilayer and to consist of a transmembrane helix (helix-1) connected via a flexible linker region, including a Glu-Tyr-Arg (EYR) motif, with a second helix (helix-2) residing with its helix long axis on the bilayer surface. Repeated molecular dynamics simulations show that Glu-28 is involved in salt bridge formation with Lys-31 and Arg-34 establishing a kink between the two helices. Helix-2 remains in a helical conformation indicating its stability and function as a “peptide float,” separating helix-1 from the rest of the protein. This leads to the conclusion that Vpu consists of three functional modules: helix-1, helix-2, and the remaining residues toward the C-terminal end.

High risk type of Human papillomavirus type 16 (HPV 16) is the causative agent of precancerous lesions that leads to cervical cancer. Cellular transformation is caused by the early (E) oncogenes named E5, E6 & E7, out of which E5 is the least understood. The E5 protein is active during viral infection. E5 protein when assembled into a hexamer allows the passage of small molecules or ions to pass through the pore.A docking approach of E5 with its three transmembrane domains (TMDs) has been used to generate a bundle model. Molecular dynamics (MD) simulations of the bundle embedded in a fully hydrated lipid bilayer allow water pockets to be generated due to the hydrophilic residues at the C-terminal side of the pore-lining second TMD. These residues are responsible to draw water molecules into the pore and thus allow easy access of ions to pass through the pore. A Full Correlation Analysis (FCA) suggests asymmetric dynamic of the helices within the monomers of the hexameric bundle. The number of Cl-ions passing through the pore is higher than that of the Na-ions under various voltage conditions. Potential of mean force (PMF) calculations have been done for a series of physiological ions crossing the pore to identify the specific selectivity of the bundle. In open pore structure ions experience almost undiscriminating potentials, while in a narrow pore Ca- and Cl-ions face large energy barriers.

Display Omitted Binding poses of antivirals to monomers and hexameric bundles of p7 of HCV.Guanidine based ligands bind better than derivatives of adamantanes and iminosugars.Binding of the best candidates is at the site of the loop.Hydrogen bonding and hydrophobic interactions are important. 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.

Chemokine CXCL-8 plays a central role in human immune response by binding to and activate its cognate receptor CXCR1, a member of the G-protein coupled receptor (GPCR) family. The full-length structure of CXCR1 is modeled by combining the structures of previous NMR experiments with those from homology modeling. Molecular docking is performed to search favorable binding sites of monomeric and dimeric CXCL-8 with CXCR1 and a mutated form of it. The receptor-ligand complex is embedded into a lipid bilayer and used in multi ns molecular dynamics (MD) simulations. A multi-steps binding mode is proposed: (i) the N-loop of CXCL-8 initially binds to the N-terminal domain of receptor CXCR1 driven predominantly by electrostatic interactions; (ii) hydrophobic interactions allow the N-terminal Glu-Leu-Arg (ELR) motif of CXCL-8 to move closer to the extracellular loops of CXCR1; (iii) electrostatic interactions finally dominate the interaction between the N-terminal ELR motif of CXCL-8 and the EC-loops of CXCR1. Mutation of CXCR1 abrogates this mode of binding. The detailed binding process may help to facilitate the discovery of agonists and antagonists for rational drug design.

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 (Ca2+) 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. Proteins 2015; 83:300–308. © 2014 Wiley Periodicals, Inc.

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelial cells leading to precancerous lesions. The HPV genome encodes three oncoproteins: E5, E6 and E7. E6 and E7 are the main transforming proteins of HPV, which are studied very well but the role of E5 is poorly understood. E5 of HPV-16, one of the “high risk” types of HPV strains, is an 83 amino acid hydrophobic membrane protein, with three hydrophobic transmembrane domains (TMDs) and short regions at the N and C termini that extend beyond the lipid bilayer. It oligomerizes into dimers which form channels most likely as hexameric bundles.The three transmembrane domains of E5 are identified using secondary structure prediction programs. These TMDs are assembled into a monomer by a ‘concerted’ docking approach in which the conformational space of the three helices is screened by simultaneously altering distance, tilt and rotational angle between them. In a consequent step loops linking the three helices are added using the program Loopy. Finally three monomers are assembled into a hexameric bundle. The bundle with TMD2 lining the pore remains intact when inserted into a hydrated lipid bilayer. It forms an almost fully water filled pore during 100 ns MD simulations. The pore of the bundle is mostly mantled by hydrophilic residues and the pore diameter is consisted with the experimental findings.