Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.     

The determinants of stability in the human prion protein: insights into folding and misfolding from the analysis of the change in the stabilization energy distribution in different conditions

The dynamic evolution of the PrP(C) from its NMR-derived conformation to a beta-sheet-rich, aggregation-prone conformation is studied through all-atom, explicit solvent molecular dynamics in different temperature and pH conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach aimed at identifying the key residues for the stabilization and folding of the protein. It is shown that under native conditions the stabilization energy is concentrated in regions of the helices H1 and H3, whereas under misfolding conditions (low pH, high temperature, or mutations in selected sites) it is spread out over helix H2. Misfolding appears to be a rearrangement of the chain that disrupts most of the native secondary structure of the protein, producing some beta-rich conformations with an energy distribution similar to that of the native state.     

The pH dependence of flavivirus envelope protein structure: insights from molecular dynamics simulations

The flavivirus membrane fusion is triggered by the acid pH of the endosomes after virus endocytosis. The proposed mechanism involves changes in the protonation state of conserved histidine residues of the E protein present in the viral surface that undergoes a series of structural rearrangements that result in the fusion between the endosome and viral bilayers. We studied the pH dependence of E protein rearrangements of dengue virus type 2, used as a model, in the pH range experimented by the virus along the fusion process. We employed a low computational cost scheme to explore the behavior of the E protein by molecular dynamics (MD) simulations of complete systems that include the protein, the solvent, and ions. The procedure alternates cyclically the update of the ionization states of the protein residues with common MD steps applied to the new ionization configuration. Important pH-dependent protein structure rearrangements consistent with the changes of the protonation states of conserved histidine residues were observed. The involvement of other conserved residues in the flavivirus in the rearrangements was also identified. The results show interesting correlations with a proposed model for the fusion mechanism, as well as the experimentally identified key residues, contributing to a better understanding of the structural changes in protein E that lead to the fusion process.     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Effects of ligand binding on the dynamics of rice nonspecific lipid transfer protein 1: a model from molecular simulations

Plant nonspecific lipid transfer proteins (nsLTPs) are small, basic proteins constituted mainly of alpha-helices and stabilized by four conserved disulfide bridges. They are characterized by the presence of a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers in vitro. In this study, molecular dynamics (MD) simulations were performed at room temperature to investigate the effects of lipid binding on the dynamic properties of rice nsLTP1. Rice nsLTP1, either in the free form or complexed with one or two lipids was subjected to MD simulations. The C-terminal loop was very flexible both before and after lipid binding, as revealed by calculating the root-mean-square fluctuation. After lipid binding, the flexibility of some residues that were not in direct contact with lipid molecules increased significantly, indicating an increase of entropy in the region distal from the binding site. Essential dynamics analysis revealed clear differences in motion between unliganded and liganded rice nsLTP1s. In the free form of rice nsLTP1, loop1 exhibited the largest directional motion. This specific essential motion mode diminished after binding one or two lipid molecules. To verify the origin of the essential motion observed in the free form of rice nsLTP1, we performed multiple sequence alignments to probe the intrinsic motion encoded in the primary sequence. We found that the amino acid sequence of loop1 is highly conserved among plant nsLTP1s, thus revealing its functional importance during evolution. Furthermore, the sequence of loop1 is composed mainly of amino acids with short side chains. In this study, we show that MD simulations, together with essential dynamics analysis, can be used to determine structural and dynamic differences of rice nsLTP1 upon lipid binding.     

Sequence dependence of amyloid fibril formation: insights from molecular dynamics simulations

The clarification of the physico-chemical determinants underlying amyloid deposition is critical for our understanding of misfolding diseases. With this purpose we have performed a systematic all-atom molecular dynamics (MD) study of a series of single point mutants of the de novo designed amyloidogenic peptide STVIIE. Sixteen different 50ns long simulations using explicit solvent have been carried out starting from four different conformations of a polymeric six-stranded beta-sheet. The simulations have provided evidence for the influence of a small number of site-specific hydrophobic interactions on the packing and stabilization of nascent aggregates, as well as the interplay between side-chain interactions and the net charge of the molecule on the strand arrangement of polymeric beta-sheets. This MD analysis has also shed light into the origin of the position dependence on mutation of beta-sheet polymerization that was found experimentally for this model system. Our results suggest that MD can be applied to detect critical positions for beta-sheet aggregation within a given amyloidogenic stretch. Studies similar to the one presented here can guide site-directed mutations or the design of drugs that specifically disrupt the key stabilizing interactions of beta-sheet aggregates.     

Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form

We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrP^Sc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrP^C source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.     

Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis

The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β‐state oligomers. Herein, we demonstrate that β‐state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full‐length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP‐induced neurotoxicity. We have characterized protein misfolding cyclic amplification‐induced monomer‐to‐oligomer conversion of PrP from three species using western blotting, circular dichroism, size‐exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β‐oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP^C in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer‐induced apoptosis via regulation of Bcl‐2, Bax, and caspase‐3 in both wild‐type and PrP^?/? cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain.

     

Molecular dynamics study on the stability of wild-type and the R220K mutant of human prion protein

Prion diseases are invariably fatal and highly infectious neurodegenerative diseases related to the structure transition of α-helix into β-sheet. In order to gain more direct insight into the molecular basis of the disease, the stability of the wild-type human prion protein (hPrPc) and the R220K mutant (m-hPrPc) was studied by molecular dynamics (MD) and flow MD simulation. Both the thermodynamic stability and the mechanical properties of hPrPc were investigated in this work. It was found that β-sheet was more readily to be unfolded in m-hPrPc. In the case of hPrPc, less content of helix was preserved after water turbulence. The H-bond network formed by the mutation-related residue 220 was found to play a key role in the stability of hPrPc.     

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt‐Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT‐TTR and L55P‐TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P‐TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single α‐helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation‐prone conformations characterized by full displacement of strands C and D from the main β‐sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H‐bond network and consequent destabilization of the CBEF β‐sheet of the β‐sandwich; (v) WT forms aggregation‐compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P‐TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.     

Assembly of a tetrameric alpha-helical bundle: computer simulations on an intermediate-resolution protein model.

Discontinuous molecular dynamics (DMD) simulation on an intermediate-resolution protein model is used to study the folding of an isolated, small model peptide to an amphipathic alpha-helix and the assembly of four of these model peptides into a four-helix bundle. A total of 129 simulations were performed on the isolated peptide, and 50 simulations were performed on the four-peptide system. Simulations efficiently sample conformational space allowing complete folding trajectories from random initial configurations to be observed within 15 min for the one-peptide system and within 15 h for the four-peptide system on a 500-MHz workstation. The native structures of both the alpha-helix and the four-helix bundle are consistent with experimental characterization studies and with results from previous simulations on these model peptides. In both the one- and four-peptide systems, the native state is achieved during simulations within an optimal temperature range, a phenomenon also observed experimentally. The ease with which our simulations yield reasonable estimates of folded structures demonstrates the power of the intermediate-resolution model developed for this work and the DMD algorithm and suggests that simulations of very long times and of multiprotein systems may be possible with this model.     

Sequence-dependent dynamical instability of the human prion protein: a comparative simulation study

The present study aimed to explore the most probable regions of the human prion protein backbone for which the initial steps of conformational transitions as a result of intrinsic and extrinsic perturbing factors on the protein structure can be assigned. A total of 0.3-μs molecular dynamics simulations on several analog structures of the protein have been performed. To mimic the impact of the extrinsic and intrinsic destructive parameters on the dynamical characteristics of the protein, mild acidic conditions and R208H mutation have been simulated. The findings indicated that distribution of conformational flexibilities along the protein chain was almost independent of the induced perturbing factors, and was mostly centralized on certain distinct parts of the structure comprising residues 132–145 and 187–203. Analyses also revealed that the segment comprising residues 187–203 may be considered as a peptide sequence, possessing high potential to start the initial steps of conformational rearrangements due to the induced physicochemical alterations. Sequence alignment and molecular dynamics data also revealed that segment 178–203 prefers to accommodate in extended structures rather than α-helices. Region 178–203 may be considered as a peptide switch capable of initiating the conformational transitions due to the introduced modifications and perturbing parameters.     

Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane β-barrel containing a central α-helix. The role of this α-helix, and of the conformational dynamics of the β-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane β-barrel is stable even in the absence of the α-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a ‘plug’. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Studies on the structural stability of rabbit prion probed by molecular dynamics simulations of its wild-type and mutants

Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect humans and animals. Rabbits are the only mammalian species reported to be resistant to infection from prion diseases isolated from other species (Vorberg et al., 2003). Fortunately, the NMR structure of rabbit prion (124-228) (PDB entry 2FJ3), the NMR structure of rabbit prion protein mutation S173N (PDB entry 2JOH) and the NMR structure of rabbit prion protein mutation I214V (PDB entry 2JOM) were released recently. This paper studies these NMR structures by molecular dynamics simulations. Simulation results confirm the structural stability of wild-type rabbit prion, and show that the salt bridge between D177 and R163 greatly contributes to the structural stability of rabbit prion protein.     

Structure refinement of membrane proteins via molecular dynamics simulations

A refinement protocol based on physics‐based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge‐based or implicit membrane‐based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid‐facing residues. Scoring with knowledge‐based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane‐based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models.     

The mechanism of high affinity pentasaccharide binding to antithrombin,insights from Gaussian accelerated molecular dynamics simulations

Abstract

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.

Communicated by Ramaswamy H. Sarma     

N-glycosylation induced changes in tau protein dynamics reveal its role in tau misfolding and aggregation: A microsecond long molecular dynamics study

Various posttranslational modifications like hyperphosphorylation, O-GlcNAcylation, and acetylation have been attributed to induce the abnormal folding in tau protein. Recent in vitro studies revealed the possible involvement of N-glycosylation of tau protein in the abnormal folding and tau aggregation. Hence, in this study, we performed a microsecond long all atom molecular dynamics simulation to gain insights into the effects of N-glycosylation on Asn-359 residue which forms part of the microtubule binding region. Trajectory analysis of the stimulations coupled with essential dynamics and free energy landscape analysis suggested that tau, in its N-glycosylated form tends to exist in a largely folded conformation having high beta sheet propensity as compared to unmodified tau which exists in a large extended form with very less beta sheet propensity. Residue interaction network analysis of the lowest energy conformations further revealed that Phe378 and Lys353 are the functionally important residues in the peptide which helped in initiating the folding process and Phe378, Lys347, and Lys370 helped to maintain the stability of the protein in the folded state.