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.

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.

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.

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.

Correlation of biological activity with computationally derived structural features from transmembrane hetero-dimers of HIV-1 Vpu with host factors.

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.

MD simulations of the central pore of ryanodine receptors and sequence comparison with 2B protein from coxsackie virus.

The regulation of intracellular Ca(2+) triggers a multitude of vital processes in biological cells. Ca(2+) permeable ryanodine receptors (RyRs) are the biggest known ion channels and play a key role in the regulation of intracellular calcium concentrations, particularly in muscle cells. In this study, we construct a computational model of the pore region of the skeletal RyR and perform molecular dynamics (MD) simulations. The dynamics and distribution of Ca(2+) around the luminal pore entry of the RyR suggest that Ca(2+) ions are channeled to the pore entry due to the arrangement of (acidic) amino acids at the extramembrane surface of the protein. This efficient mechanism of Ca(2+) supply is thought to be part of the mechanism of Ca(2+) conductance of RyRs. Viral myocarditis is predominantly caused by coxsackie viruses that induce the expression of the protein 2B which is known to affect intracellular Ca(2+) homeostasis in infected cells. From our sequence comparison, it is hypothesized, that modulation of RyR could be due to replacement of its transmembrane domains (TMDs) by those domains of the viral channel forming protein 2B of coxsackie virus. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Viral channel proteins in intracellular protein-protein communication: Vpu of HIV-1, E5 of HPV16 and p7 of HCV.

Viral channel forming proteins are known for their capability to make the lipid membrane of the host cell and its subcellular compartments permeable to ions and small compounds. There is increasing evidence that some of the representatives of this class of proteins are also strongly interacting with host proteins and the effectiveness of this interaction seems to be high. Interaction of viral channel proteins with host factors has been proposed by bioinformatics approaches and has also been identified experimentally. An overview of the interactions with host proteins is given for Vpu from HIV-1, E5 from HPV-16 and p7 from HCV. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

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.

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.

Qualitative computational bioanalytics: assembly of viral channel-forming peptides around mono and divalent ions.

A fine-grained docking protocol was used to generate a bundle-like structure of the bitopic membrane protein Vpu from HIV-1. Vpu is a type I membrane protein with 81 amino acids. It is proposed that Vpu forms ion- and substrate-conducting bundles, which are located at the plasma membrane in the infected cell. The Vpu1-32 peptide that includes the transmembrane domain (TMD) is assembled into homo-pentameric bundles around prepositioned Na, K, Ca or Cl ions. For bundles with the lowest energy, the TMDs generate a hydrophobic pore. Bundles in which Ser-24 faces the pore have higher energy. The tilt of the helices in the lowest energy bundles is larger than bundles with serines facing the pore. Left-handed bundles are lowest in energy where the ions are located at the serines.

Assembling viral channel forming proteins: Vpu from HIV-1.

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.

Assembling an ion channel: ORF 3a from SARS-CoV.

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 + TMD3 as well as TMD2 + TMD3 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 ≈ 2 PCl ) rectifying channel. In the presence of nonphysiological concentration of Ca-ions the channel develops channel activity.

Molecular dynamics simulations and conductance studies of the interaction of VP1 N-terminus from Polio virus and gp41 fusion peptide from HIV-1 with lipid membranes.

The icosahedral Polio virus capsid consists of 60 copies of each of the coat proteins VP1, VP2, VP3 and myristolyated VP4 (myrVP4). Catalyzed by the host cell receptor the Polio virus enters the host cell via externalization of myrVP4 and the N terminal part of VP1. There are several assumptions about the individual role of both of the proteins in the mechanism of membrane attachment and genome injection. We use the first 32 N terminal amino acids of VP1 and applied molecular dynamics simulations to assess its mechanism of function when attached and inserted into hydrated lipid membranes (POPC). Helical models are placed in various positions in regard to the lipid membrane to start with. As a comparison, the first 33 amino acids of the fusion peptide of gp41 of HIV-1 are simulated under identical conditions. Computational data support the idea that VP1 is not penetrating into the membrane to form a pore; it rather lays on the membrane surface and only perturbs the membrane. Furthermore, this idea is strengthened by channel recordings of both peptides showing irregular openings.

Viral channel forming proteins - modeling the target.

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.

ORF8a of SARS-CoV forms an ion channel: experiments and molecular dynamics simulations.

ORF8a protein is 39 residues long and contains a single transmembrane domain. The protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers that forms cation-selective ion channels with a main conductance level of 8.9±0.8pS at elevated temperature (38.5°C). Computational modeling studies including multi nanosecond molecular dynamics simulations in a hydrated POPC lipid bilayer are done with a 22 amino acid transmembrane helix to predict a putative homooligomeric helical bundle model. A structural model of a pentameric bundle is proposed with cysteines, serines and threonines facing the pore.

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.

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.

Ultrasensitive in situ label-free DNA detection using a GaN nanowire-based extended-gate field-effect-transistor sensor.

In this study, we have successfully demonstrated that a GaN nanowire (GaNNW) based extended-gate field-effect-transistor (EGFET) biosensor is capable of specific DNA sequence identification under label-free in situ conditions. Our approach shows excellent integration of the wide bandgap semiconducting nature of GaN, surface-sensitivity of the NW-structure, and high transducing performance of the EGFET-design. The simple sensor-architecture, by direct assembly of as-synthesized GaNNWs with a commercial FET device, can achieve an ultrahigh detection limit below attomolar level concentrations: about 3 orders of magnitude higher in resolution than that of other FET-based DNA-sensors. Comparative in situ studies on mismatches ("hotspot" mutations related to human p53 tumor-suppressor gene) and complementary targets reveal excellent selectivity and specificity of the sensor, even in the presence of noncomplementary DNA strands, suggesting the potential pragmatic application in complex clinical samples. In comparison with GaN thin film, NW-based EGFET exhibits excellent performance with about 2 orders higher sensitivity, over a wide detection range, 10(-19)-10(-6) M, reaching about a 6-orders lower detection limit. Investigations illustrate the unique and distinguished feature of nanomaterials. Detailed studies indicate a positive effect of energy band alignment at the biomaterials-semiconductor hybrid interface influencing the effective capacitance and carrier-mobility of the system.

Structural implications of mutations assessed by molecular dynamics: Vpu1-32 from HIV-1.

Structural pore models are generated for Vpu(1-32)WT from HIV-1 as well as for three mutants W23L, S24L and R31V. A computational methodology is employed which samples the whole conformational space of the pentameric assemblies of Vpu. The analysis of the related energy landscape reveals a small set of reasonable pore models, which are thoroughly investigated regarding their structural properties as well as their putative stability under native-like conditions. The models are also discussed in respect of earlier experimental findings about their channel activities. The study proposes functional pores reflecting the experimentally found conductance states of Vpu and its mutants.