Perspective for Molecular Dynamics Simulation Studies of Amyloid-ß Aggregates.

The cause of Alzheimer's disease is related to aggregates such as oligomers and amyloid fibrils consisting of amyloid-ß (Aß) peptides. Molecular dynamics (MD) simulation studies have been conducted to understand the molecular mechanism of the formation and disruption of Aß aggregates. In this Perspective, the MD simulation studies are classified into four categories, focusing on the target systems: aggregation of Aß peptides in bulk solution, Aß aggregation at the interface, aggregation inhibitor against Aß peptides, and nonequilibrium MD simulation of Aß aggregates. MD simulation studies in these categories are first reviewed. Future perspectives in each category are then presented. Finally, the overall perspective is presented on how MD simulations of Aß aggregates can be utilized for developing Alzheimer's disease treatment.

Dissociation process of polyalanine aggregates by free electron laser irradiation.

Polyalanine (polyA) disease-causative proteins with an expansion of alanine repeats can be aggregated. Although curative treatments for polyA diseases have not been explored, the dissociation of polyA aggregates likely reduces the cytotoxicity of polyA. Mid-infrared free electron laser (FEL) successfully dissociated multiple aggregates. However, whether the FEL dissociates polyA aggregates like other aggregates has not been tested. Here, we show that FEL at 6.1 µm experimentally weakened the extent of aggregation of a peptide with 13 alanine repeats (13A), and the irradiated 13A exerted lesser cytotoxicity to neuron-like cells than non-irradiated 13A. Then, we applied molecular dynamics (MD) simulation to follow the dissociation process by FEL. We successfully observed how the intermolecular ß-sheet of polyA aggregates was dissociated and separated into monomers with helix structures upon FEL irradiation. After the dissociation by FEL, water molecules inhibited the reformation of polyA aggregates. We recently verified the same dissociation process using FEL-treated amyloid-ß aggregates. Thus, a common mechanism underlies the dissociation of different protein aggregates that cause different diseases, polyA disease and Alzheimer's disease. However, MD simulation indicated that polyA aggregates are less easily dissociated than amyloid-ß aggregates and require longer laser irradiation due to hydrophobic alanine repeats.

Effective mechanical potential of cell-cell interaction explains three-dimensional morphologies during early embryogenesis.

Mechanical forces are critical for the emergence of diverse three-dimensional morphologies of multicellular systems. However, it remains unclear what kind of mechanical parameters at cellular level substantially contribute to tissue morphologies. This is largely due to technical limitations of live measurements of cellular forces. Here we developed a framework for inferring and modeling mechanical forces of cell-cell interactions. First, by analogy to coarse-grained models in molecular and colloidal sciences, we approximated cells as particles, where mean forces (i.e. effective forces) of pairwise cell-cell interactions are considered. Then, the forces were statistically inferred by fitting the mathematical model to cell tracking data. This method was validated by using synthetic cell tracking data resembling various in vivo situations. Application of our method to the cells in the early embryos of mice and the nematode Caenorhabditis elegans revealed that cell-cell interaction forces can be written as a pairwise potential energy in a manner dependent on cell-cell distances. Importantly, the profiles of the pairwise potentials were quantitatively different among species and embryonic stages, and the quantitative differences correctly described the differences of their morphological features such as spherical vs. distorted cell aggregates, and tightly vs. non-tightly assembled aggregates. We conclude that the effective pairwise potential of cell-cell interactions is a live measurable parameter whose quantitative differences can be a parameter describing three-dimensional tissue morphologies.

The Double-Layered Structure of Amyloid-ß Assemblage on GM1-Containing Membranes Catalytically Promotes Fibrillization.

Alzheimer's disease (AD) is associated with progressive accumulation of amyloid-ß (Aß) cross-ß fibrils in the brain. Aß species tightly associated with GM1 ganglioside, a glycosphingolipid abundant in neuronal membranes, promote amyloid fibril formation; therefore, they could be attractive clinical targets. However, the active conformational state of Aß in GM1-containing lipid membranes is still unknown. The present solid-state nuclear magnetic resonance study revealed a nonfibrillar Aß assemblage characterized by a double-layered antiparallel ß-structure specifically formed on GM1 ganglioside clusters. Our data show that this unique assemblage was not transformed into fibrils on GM1-containing membranes but could promote conversion of monomeric Aß into fibrils, suggesting that a solvent-exposed hydrophobic layer provides a catalytic surface evoking Aß fibril formation. Our findings offer structural clues for designing drugs targeting catalytically active Aß conformational species for the development of anti-AD therapeutics.

Probing protein misfolding and dissociation with an infrared free-electron laser.

Misfolding is observed in the mutant proteins that are causative for neurodegenerative disorders such as polyglutamine diseases. These proteins are prone to aggregate in the cytoplasm and nucleus of cells. To reproduce cells with the aggregated proteins, gene expression system is usually applied, in which the expression construct having the mutated DNA sequence of the interest is transfected into cells. The transfected DNA is finally converted into the mutant protein, which is gradually aggregated in the cells. In addition, a simple method to prepare the cells having aggregates inside has been recently applied. Peptides were first aggregated by incubating them in water. The aggregates are spontaneously taken up by cells because aggregated proteins generally transfer between cells. Peptides with different degrees of aggregation can be made by changing the incubation times and temperatures, which enables to examine contribution of aggregation to the toxicity to the recipient cells. Moreover, such cells can be used for therapeutic researches of diseases in which aggregates are involved. In this chapter, we show methods to induce aggregation of peptides. The functional analyses of the cells with aggregates are also described. Then, experimental dissociation of the aggregates produced using this method by mid infrared free electron laser irradiation and its theoretical support by molecular dynamics simulation are introduced as the therapeutic research for neurodegenerative disorders.

Ingenuity in performing replica permutation: How to order the state labels for improving sampling efficiency.

In the replica-permutation method, an advanced version of the replica-exchange method, all combinations of replicas and parameters are considered for parameter permutation, and a list of all the combinations is prepared. Here, we report that the temperature transition probability depends on how the list is created, especially in replica permutation with solute tempering (RPST). We found that the transition probabilities decrease at large replica indices when the combinations are sequentially assigned to the state labels as in the originally proposed list. To solve this problem, we propose to modify the list by randomly assigning the combinations to the state labels. We performed molecular dynamics simulations of amyloid-ß(16-22) peptides using RPST with the "randomly assigned" list (RPST-RA) and RPST with the "sequentially assigned" list (RPST-SA). The results show the decreases in the transition probabilities in RPST-SA are eliminated, and the sampling efficiency is improved in RPST-RA.

Inhibition of amyloid-ß(16-22) aggregation by polyphenols using replica permutation with solute tempering molecular dynamics simulation.

Aggregates of amyloid-ß (Aß) peptides are thought to cause Alzheimer's disease. Polyphenolic compounds are known to inhibit Aß aggregation. We applied replica permutation with solute tempering (RPST) to the system of Aß fragments, Aß(16-22), and polyphenols to elucidate the mechanism of inhibition of Aß aggregation. The RPST molecular dynamics simulations were performed for two polyphenols, myricetin (MYC) and rosmarinic acid (ROA). Two Aß fragments were distant, and the number of residues forming the intermolecular ß-sheet was reduced in the presence of MYC and ROA compared with that in the absence of polyphenols. MYC was found to interact with glutamic acid and phenylalanine of Aß fragments. These interactions induce helix structure formation of Aß fragments, making it difficult to form ß-sheet. ROA interacted with glutamic acid and lysine, which reduced the hydrophilic interaction between Aß fragments. These results indicate that these polyphenols inhibit the aggregation of Aß fragments with different mechanisms.

Key Residue for Aggregation of Amyloid-ß Peptides.

It is known that oligomers of amyloid-ß (Aß) peptide are associated with Alzheimer's disease. Aß has two isoforms: Aß40 and Aß42. Although the difference between Aß40 and Aß42 is only two additional C-terminal residues, Aß42 aggregates much faster than Aß40. It is unknown what role the C-terminal two residues play in accelerating aggregation. Since Aß42 is more toxic than Aß40, its oligomerization process needs to be clarified. Moreover, clarifying the differences between the oligomerization processes of Aß40 and Aß42 is essential to elucidate the key factors of oligomerization. Therefore, to investigate the dimerization process, which is the early oligomerization process, Hamiltonian replica-permutation molecular dynamics simulations were performed for Aß40 and Aß42. We identified a key residue, Arg5, for the Aß42 dimerization. The two additional residues in Aß42 allow the C-terminus to form contact with Arg5 because of the electrostatic attraction between them, and this contact stabilizes the ß-hairpin. This ß-hairpin promotes dimer formation through the intermolecular ß-bridges. Thus, we examined the effects of amino acid substitutions of Arg5, thereby confirming that the mutations remarkably suppressed the aggregation of Aß42. Moreover, the mutations of Arg5 suppressed the Aß40 aggregation. It was found by analyzing the simulations that Arg5 is important for Aß40 to form intermolecular contacts. Thus, it was clarified that the role of Arg5 in the oligomerization process varies due to the two additional C-terminal residues.

Application study of infrared free-electron lasers towards the development of amyloidosis therapy.

Amyloidosis is known to be caused by the deposition of amyloid fibrils into various biological tissues; effective treatments for the disease are little established today. An infrared free-electron laser (IR-FEL) is an accelerator-based picosecond-pulse laser having tunable infrared wavelengths. In the current study, the irradiation effect of an IR-FEL was tested on an 11-residue peptide (NFLNCYVSGFH) fibril from ß2-microglobulin (ß2M) with the aim of applying IR-FELs to amyloidosis therapy. Infrared microspectroscopy (IRM) and scanning electron microscopy showed that a fibril of ß2M peptide was clearly dissociated by IR-FEL at 6.1 µm (amide I) accompanied by a decrease of the ß-sheet and an increase of the α-helix. No dissociative process was recognized at 6.5 µm (amide II) as well as at 5.0 µm (non-specific wavelength). Equilibrium molecular dynamics simulations indicated that the α-helix can exist stably and the probability of forming interchain hydrogen bonds associated with the internal asparagine residue (N4) is notably reduced compared with other amino acids after the ß-sheet is dissociated by amide I specific irradiation. This result implies that N4 plays a key role for recombination of hydrogen bonds in the dissociation of the ß2M fibril. In addition, the ß-sheet was disrupted at temperatures higher than 340 K while the α-helix did not appear even though the fibril was heated up to 363 K as revealed by IRM. The current study gives solid evidence for the laser-mediated conversion from ß-sheet to α-helix in amyloid fibrils at the molecular level.

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2.

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a "bucket brigade." This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.

Molecular dynamics simulations of amyloid-ß peptides in heterogeneous environments.

Alzheimer's disease is thought to be caused by the aggregation of amyloid-ß (Aß) peptides. Their aggregation is accelerated at hydrophilic/hydrophobic interfaces such as the air-water interface and the surface of monosialotetrahexosylganglioside (GM1) clusters on neuronal cell membranes. In this review, we present recent studies of full-length Aß (Aß40) peptides and Aß(16-22) fragments in such heterogeneous environments by molecular dynamics (MD) simulations. These peptides have both hydrophilic and hydrophobic amino-acid residues and tend to exist at the hydrophilic/hydrophobic interface. Therefore, the peptide concentration increases at the interface, which is one of the factors that promote aggregation. Furthermore, it was found that Aß40 forms an α-helix structure and then a ß-hairpin structure at the interface. The ß-hairpin promotes the formation of oligomers with intermolecular ß-sheets. It means that not only the high concentration of Aß40 at the interface but also the structure of Aß40 itself promotes aggregation. In addition, MD simulations of Aß40 on recently-developed GM1-glycan clusters showed that the HHQ (13-15) segment of Aß40 is important for the recognition of GM1-glycan clusters. It was also elucidated that Aß40 forms a helix structure in the C-terminal region on the GM1-glycan cluster. This result suggests that the helix formation, which is the first step in the conformational changes toward pathological aggregation, is initiated at the GM1-glycan moieties rather than at the lipid-ceramide moieties. These studies will enhance the physicochemical understanding of the structural changes of Aß at the heterogeneous interfaces and the mechanism of Alzheimer's disease pathogenesis.

Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP2 regulation.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel's pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein-ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.

Molecular Dynamics Simulation Studies on the Aggregation of Amyloid-ß Peptides and Their Disaggregation by Ultrasonic Wave and Infrared Laser Irradiation.

Alzheimer's disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-ß (Aß) peptides. This review article presents molecular dynamics (MD) simulation studies of Aß peptides and Aß fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, and disruption of the amyloid fibril by ultrasonic wave and infrared laser irradiation. In the aggregation of Aß, a ß-hairpin structure promotes the formation of intermolecular ß-sheet structures. Aß peptides tend to exist at hydrophilic/hydrophobic interfaces and form more ß-hairpin structures than in bulk water. These facts are the reasons why the aggregation is accelerated at the interface. We also explain how polyphenols, which are attracting attention as aggregation inhibitors of Aß peptides, interact with Aß. An MD simulation study of the Aß amyloid fibrils in equilibrium is also presented: the Aß amyloid fibril has a different structure at one end from that at the other end. The amyloid fibrils can be destroyed by ultrasonic wave and infrared laser irradiation. The molecular mechanisms of these amyloid fibril disruptions are also explained, particularly focusing on the function of water molecules. Finally, we discuss the prospects for developing treatments for Alzheimer's disease using MD simulations.

Replica permutation with solute tempering for molecular dynamics simulation and its application to the dimerization of amyloid-ß fragments.

We propose the replica permutation with solute tempering (RPST) by combining the replica-permutation method (RPM) and the replica exchange with solute tempering (REST). Temperature permutations are performed among more than two replicas in RPM, whereas temperature exchanges are performed between two replicas in the replica-exchange method (REM). The temperature transition in RPM occurs more efficiently than in REM. In REST, only the temperatures of the solute region, the solute temperatures, are exchanged to reduce the number of replicas compared to REM. Therefore, RPST is expected to be an improved method taking advantage of these methods. For comparison, we applied RPST, REST, RPM, and REM to two amyloid-ß(16-22) peptides in explicit water. We calculated the transition ratio and the number of tunneling events in the temperature space and the number of dimerization events of amyloid-ß(16-22) peptides. The results indicate that, in RPST, the number of replicas necessary for frequent random walks in the temperature and conformational spaces is reduced compared to the other three methods. In addition, we focused on the dimerization process of amyloid-ß(16-22) peptides. The RPST simulation with a relatively small number of replicas shows that the two amyloid-ß(16-22) peptides form the intermolecular antiparallel ß-bridges due to the hydrophilic side-chain contact between Lys and Glu and hydrophobic side-chain contact between Leu, Val, and Phe, which stabilizes the dimer of the peptides.

Tardigrade Secretory-Abundant Heat-Soluble Protein Varies Entrance Propensity Depending on the Amino-Acid Sequence.

Secretory-abundant heat-soluble (SAHS) proteins, which constitute a protein family unique to tardigrades, are thought to be essential for anhydrobiosis. Our previous study has revealed that one of the SAHS proteins of Ramazzottius varieornatus (RvSAHS1) has a more flexible entrance than a mammalian fatty-acid-binding protein, which has a crystal structure similar to that of RvSAHS1. Recently, SAHS paralogs that are expressed abundantly and specifically in the early embryos of this tardigrade and Hypsibius exemplaris have been identified. Comparing these amino-acid sequences with that of RvSAHS1, we have found characteristic differences as I113F and D146T. In this study, we investigate I113F and D146T mutants' properties of RvSAHS1 using molecular dynamics simulations and compare the structures and fluctuations of their entrances with those of the wild type. The two mutants exhibit different properties at the entrance of the ß-barrel structure. The I113F mutant tends to close the entrance more than the wild type due to the enhanced hydrophobic network inside the cavity. The D146T mutant, in contrast to the I113F mutant, tends to open the entrance. The mechanism by which this mutation opens the entrance is also discussed. Even though only a single mutation located far from the entrance is added to the wild type, there is a clear difference in the tendency to open and close the ß-barrel entrance. It indicates that the entrance properties of the SAHS protein are sensitive to the amino-acid sequence.

All-Atom Molecular Dynamics Simulation Methods for the Aggregation of Protein and Peptides: Replica Exchange/Permutation and Nonequilibrium Simulations.

Protein aggregates are associated with more than 40 serious human diseases. To understand the formation mechanism of protein aggregates at atomic level, all-atom molecular dynamics (MD) simulation is a powerful computational tool. In this chapter, we review the all-atom MD simulation methods that are useful for study on the protein aggregation. We first explain conventional MD simulation methods in physical statistical ensembles, such as the canonical and isothermal-isobaric ensembles. We then describe the generalized-ensemble algorithms such as replica-exchange and replica-permutation MD methods. These methods can overcome a difficulty, in which simulations tend to get trapped in local-minimum free-energy states. Finally we explain the nonequilibrium MD method. Some simulation results based on these methods are also presented.

"Bucket brigade" using lysine residues in RNA-dependent RNA polymerase of SARS-CoV-2.

The RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a promising drug target for coronavirus disease 2019 (COVID-19) because it plays the most important role in the replication of the RNA genome. Nucleotide analogs such as remdesivir and favipiravir are thought to interfere with the RNA replication by RdRp. More specifically, they are expected to compete with nucleoside triphosphates, such as ATP. However, the process in which these drug molecules and nucleoside triphosphates are taken up by RdRp remains unknown. In this study, we performed all-atom molecular dynamics simulations to clarify the recognition mechanism of RdRp for these drug molecules and ATP that were at a distance. The ligand recognition ability of RdRp decreased in the order of remdesivir, favipiravir, and ATP. We also identified six recognition paths. Three of them were commonly found in all ligands, and the remaining three paths were ligand-dependent ones. In the common two paths, it was observed that the multiple lysine residues of RdRp carried the ligands to the binding site like a "bucket brigade." In the remaining common path, the ligands directly reached the binding site. Our findings contribute to the understanding of the efficient ligand recognition by RdRp at the atomic level.

Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible ß-Barrel Structure in Solution and Keeps This Structure in Dehydration.

Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the ßD and ßE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the ß-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

Dynamic properties of SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases studied by molecular dynamics simulations.

One of the promising drug targets against COVID-19 is an RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. The tertiary structures of the SARS-CoV-2 and SARS-CoV RdRps are almost the same. However, the RNA-synthesizing activity of the SARS-CoV RdRp is higher than that of the SARS-CoV-2 RdRp. We performed molecular dynamics simulations and found differences in their dynamic properties. In the SARS-CoV RdRp, motifs A-G, which form the active site, are up to 63% closer to each other. We also observed cooperative domain motion in the SARS-CoV RdRp. Such dynamic differences may cause the activity differences between the two RdRps.