Cysteine Mutations in the Ebolavirus Matrix Protein VP40 Promote Phosphatidylserine Binding by Increasing the Flexibility of a Lipid-Binding Loop.

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS.

Conformational Flexibility of the Protein-Protein Interfaces of the Ebola Virus VP40 Structural Matrix Filament.

The Ebola virus (EBOV) is a virulent pathogen that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV transformer protein VP40 plays crucial roles in viral assembly and budding at the plasma membrane of infected cells. One of VP40's roles is to form the long, flexible, pleomorphic filamentous structural matrix for the virus. Each filament contains three unique interfaces: monomer NTD-NTD to form a dimer, dimer-to-dimer NTD-NTD oligomerization to form a hexamer, and end-to-end hexamer CTD-CTD to build the filament. However, the atomic-level details of conformational flexibility of the VP40 filament are still elusive. In this study, we have performed explicit-solvent, all-atom molecular dynamic simulations to explore the conformational flexibility of the three different interface structures of the filament. Using dynamic network analysis and other calculational methods, we find that the CTD-CTD hexamer interface with weak interdomain amino acid communities is the most flexible, and the NTD-NTD oligomer interface with strong interdomain communities is the least flexible. Our study suggests that the high flexibility of the CTD-CTD interface may be essential for the supple bending of the Ebola filovirus, and such flexibility may present a target for molecular interventions to disrupt the Ebola virus functioning.

Molecular mechanisms of pore formation and membrane disruption by the antimicrobial lantibiotic peptide Mutacin 1140.

The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane. Our results suggest that MU1140-lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140-lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.

A cylindrical assembly model and dynamics of the Ebola virus VP40 structural matrix.

The Ebola filovirus causes severe hemorrhagic fever with a high fatality rate in humans. The primary structural matrix protein VP40 displays transformer-protein characteristics and exists in different conformational and oligomeric states. VP40 plays crucial roles in viral assembly and budding at the plasma membrane of the infected cells and is capable of forming virus-like particles without the need for other Ebola proteins. However, no experimental three-dimensional structure for any filovirus VP40 cylindrical assembly matrix is currently available. Here, we use a protein-protein docking approach to develop cylindrical assembly models for an Ebola virion and also for a smaller structural matrix that does not contain genetic material. These models match well with the 2D averages of cryo-electron tomograms of the authentic virion. We also used all-atom molecular dynamics simulations to investigate the stability and dynamics of the cylindrical models and the interactions between the side-by-side hexamers to determine the amino acid residues that are especially important for stabilizing the hexamers in the cylindrical ring configuration matrix assembly. Our models provide helpful information to better understand the assembly processes of filoviruses and such structural studies may also lead to the design and development of antiviral drugs.

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments.

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle.

The Ebola virus protein VP40 hexamer enhances the clustering of PI(4,5)P2 lipids in the plasma membrane.

The Ebola virus is a lipid-enveloped virus that obtains its lipid coat from the plasma membrane of the host cell it infects during the budding process. The Ebola virus protein VP40 localizes to the inner leaflet of the plasma membrane and forms the viral matrix, which provides the major structure for the Ebola virus particles. VP40 is initially a dimer that rearranges to a hexameric structure that mediates budding. VP40 hexamers and larger filaments have been shown to be stabilized by PI(4,5)P2 in the plasma membrane inner leaflet. Reduction in the plasma membrane levels of PI(4,5)P2 significantly reduce formation of VP40 oligomers and virus-like particles. We investigated the lipid-protein interactions in VP40 hexamers at the plasma membrane. We quantified lipid-lipid self-clustering by calculating the fractional interaction matrix and found that the VP40 hexamer significantly enhances the PI(4,5)P2 clustering. The radial pair distribution functions suggest a strong interaction between PI(4,5)P2 and the VP40 hexamer. The cationic Lys side chains are found to mediate the PIP2 clustering around the protein, with cholesterol filling the space between the interacting PIP2 molecules. These computational studies support recent experimental data and provide new insights into the mechanisms by which VP40 assembles at the plasma membrane inner leaflet, alters membrane curvature, and forms new virus-like particles.

Structural propensities and entropy effects in peptide helix-coil transitions.

The helix-coil transition in peptides is a critical structural transition leading to functioning proteins. Peptide chains have a large number of possible configurations that must be accounted for in statistical mechanical investigations. Using hydrogen bond and local helix propensity interaction terms, we develop a method for obtaining and incorporating the degeneracy factor that allows the exact calculation of the partition function for a peptide as a function of chain length. The partition function is used in calculations for engineered peptide chains of various lengths that allow comparison with a variety of different types of experimentally measured quantities, such as fraction of helicity as a function of both temperature and chain length, heat capacity, and denaturation studies. When experimental sensitivity in helicity measurements is properly accounted for in the calculations, the calculated curves fit well with the experimental curves. We determine values of interaction energies for comparison with known biochemical interactions, as well as quantify the difference in the number of configurations available to an amino acid in a random coil configuration compared to a helical configuration.

Lattice model simulations of the effects of the position of a peptide trigger segment on helix folding and dimerization.

The folding and dimerization of proteins is greatly facilitated by the presence of a trigger site, a segment of amino acids that has a higher propensity for forming α-helix structure as compared to the rest of the chain. In addition to the helical propensity of each chain, dimerization can also be facilitated by interhelical interactions such as saltbridges, and interfacial contacts of different strengths. In this work, we are interested in understanding the interplay of these interactions in a model peptide system. We investigate how these different interactions influence the kinetics of dimer formation and the stability of the fully formed dimer. We use lattice model computer simulations to investigate how the effectiveness of the trigger segment and its saltbridges depends on the location along the protein primary sequence. For different positions of the trigger segment, heat capacity and free energy of unfolded and folded configurations are calculated to study the thermodynamics of folding and dimerization. The kinetics of the process is investigated by calculating characteristic folding times. The thermodynamic and kinetic data from the simulations combine to show that the dimerization process of the model system is faster when the segment with high helical propensity is located near either end of the peptide, as compared to the middle of the chain. The dependence of the stability of the dimer on the trigger segment's position is also studied. The stability can play a role in the ability of the dimer to perform a biological function that involves partial unzipping. The results on folding and dimer stability provide important insights for designing proteins that involve trigger sites.

Lattice model simulation of interchain protein interactions and the folding dynamics and dimerization of the GCN4 Leucine zipper.

The highest level in the hierarchy of protein structure and folding is the formation of protein complexes through protein-protein interactions. We have made modifications to a well established computer lattice model to expand its applicability to two-protein dimerization and aggregation. Based on Brownian dynamics, we implement translation and rotation moves of two peptide chains relative to each other, in addition to the intrachain motions already present in the model. We use this two-chain model to study the folding dynamics of the yeast transcription factor GCN4 leucine zipper. The calculated heat capacity curves agree well with experimental measurements. Free energy landscapes and median first passage times for the folding process are calculated and elucidate experimentally measured characteristics such as the multistate nature of the dimerization process.

Sampling of states for estimating the folding funnel entropy and energy landscape of a model alpha-helical hairpin peptide.

Protein folding times are many orders of magnitude shorter than would occur if the peptide chain randomly sampled possible configurations, which implies that protein folding is a directed process. The detailed shape of protein's energy landscape determines the rate and reliability of folding to the native state, but the large number of structural degrees of freedom generates an energy landscape that is hard to visualize because of its high dimensionality. A commonly used picture is that of an energy funnel leading from high energy random coil state down to the low energy native state. As lattice computer models of protein dynamics become more realistic, the number of possible configurations becomes too large to count directly. Statistical mechanic and thermodynamic approaches allow us to count states in an approximate manner to quantify the entropy and energy of the energy landscape within a folding funnel for an alpha-helical protein. We also discuss the problems that arise in attempting to count the huge number of individual states of the random coil at the top of the funnel.

Removal of kinetic traps and enhanced protein folding by strategic substitution of amino acids in a model alpha-helical hairpin peptide.

The presence of non-native kinetic traps in the free energy landscape of a protein may significantly lengthen the overall folding time so that the folding process becomes unreliable. We use a computational model alpha-helical hairpin peptide to calculate structural free energy landscapes and relate them to the kinetics of folding. We show how protein engineering through strategic changes in only a few amino acid residues along the primary sequence can greatly increase the speed and reliability of the folding process, as seen experimentally. These strategic substitutions also prevent the formation of long-lived misfolded configurations that can cause unwanted aggregations of peptides. These results support arguments that removal of kinetic traps, obligatory or nonobligatory, is crucial for fast folding.