Molecular dynamics simulations reveal the importance of amyloid-beta oligomer ß-sheet edge conformations in membrane permeabilization.

Oligomeric aggregates of the amyloid-beta peptide(1-42) (Aß42) are regarded as a primary cause of cytotoxicity related to membrane damage in Alzheimer's disease. However, a dynamical and structural characterization of pore-forming Aß42 oligomers at atomic detail has not been feasible. Here, we used Aß42 oligomer structures previously determined in a membrane-mimicking environment as putative model systems to study the pore formation process in phospholipid bilayers with all-atom molecular dynamics simulations. Multiple Aß42 oligomer sizes, conformations, and N-terminally truncated isoforms were investigated on the multi-µs time scale. We found that pore formation and ion permeation occur via edge conductivity and exclusively for ß-sandwich structures that feature exposed side-by-side ß-strand pairs formed by residues 9 to 21 of Aß42. The extent of pore formation and ion permeation depends on the insertion depth of hydrophilic residues 13 to 16 (HHQK domain) and thus on subtle differences in the overall stability, orientation, and conformation of the aggregates in the membrane. Additionally, we determined that backbone carbonyl and polar side-chain atoms from the edge strands directly contribute to the coordination sphere of the permeating ions. Furthermore, point mutations that alter the number of favorable side-chain contacts correlate with the ability of the Aß42 oligomer models to facilitate ion permeation in the bilayer center. Our findings suggest that membrane-inserted, layered ß-sheet edges are a key structural motif in pore-forming Aß42 oligomers independent of their size and play a pivotal role in aggregate-induced membrane permeabilization.

Biomolecular simulations at the exascale: From drug design to organelles and beyond.

The rapid advancement in computational power available for research offers to bring not only quantitative improvements, but also qualitative changes in the field of biomolecular simulation. Here, we review the state of biomolecular dynamics simulations at the threshold to exascale resources becoming available. Both developments in parallel and distributed computing will be discussed, providing a perspective on the state of the art of both. A main focus will be on obtaining binding and conformational free energies, with an outlook to macromolecular complexes and (sub)cellular assemblies.

The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils.

Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two ß-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well.

The 3D structure of lipidic fibrils of α-synuclein.

α-synuclein misfolding and aggregation into fibrils is a common feature of α-synucleinopathies, such as Parkinson's disease, in which α-synuclein fibrils are a characteristic hallmark of neuronal inclusions called Lewy bodies. Studies on the composition of Lewy bodies extracted postmortem from brain tissue of Parkinson's patients revealed that lipids and membranous organelles are also a significant component. Interactions between α-synuclein and lipids have been previously identified as relevant for Parkinson's disease pathology, however molecular insights into their interactions have remained elusive. Here we present cryo-electron microscopy structures of six α-synuclein fibrils in complex with lipids, revealing specific lipid-fibril interactions. We observe that phospholipids promote an alternative protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities of the fibrils. Together with our previous studies, these structures also indicate a mechanism for fibril-induced lipid extraction, which is likely to be involved in the development of α-synucleinopathies. Specifically, one potential mechanism for the cellular toxicity is the disruption of intracellular vesicles mediated by fibrils and oligomers, and therefore the modulation of these interactions may provide a promising strategy for future therapeutic interventions.

Secondary structure propensities in peptide folding simulations: a systematic comparison of molecular mechanics interaction schemes.

We present a systematic study directed toward the secondary structure propensity and sampling behavior in peptide folding simulations with eight different molecular dynamics force-field variants in explicit solvent. We report on the combinational result of force field, water model, and electrostatic interaction schemes and compare to available experimental characterization of five studied model peptides in terms of reproduced structure and dynamics. The total simulation time exceeded 18 mus and included simulations that started from both folded and extended conformations. Despite remaining sampling issues, a number of distinct trends in the folding behavior of the peptides emerged. Pronounced differences in the propensity of finding prominent secondary structure motifs in the different applied force fields suggest that problems point in particular to the balance of the relative stabilities of helical and extended conformations.

Primary steps of pH-dependent insulin aggregation kinetics are governed by conformational flexibility.

Insulin aggregation critically depends on pH. The underlying energetic and structural determinants are, however, unknown. Here, we measure the kinetics of the primary aggregation steps of the insulin monomer in vitro and relate it to its conformational flexibility. To assess these primary steps the monomer concentration was monitored by mass spectrometry at various pH values and aggregation products were imaged by atomic force microscopy. Lowering the pH from 3 to 1.6 markedly accelerated the observed aggregation kinetics. The influence of pH on the monomer structure and dynamics in solution was studied by molecular dynamics simulations, with the protonation states of the titrable groups obtained from electrostatic calculations. Reduced flexibility was observed for low pH values, mainly in the C terminus and in the helix of the B chain; these corresponded to an estimated entropy loss of 150 J mol(-1) K(-1). The striking correlation between entropy loss and pH value is consistent with the observed kinetic traces. In analogy to the well-known Phi value analysis, this result allows the extraction of structural information about the rate determining transition state of the primary aggregation steps. In particular, we suggest that the residues in the helix of the B chain are involved in this transition state.

Resolving the Atomistic Modes of Anle138b Inhibitory Action on Peptide Oligomer Formation.

The diphenyl-pyrazole compound anle138b is a known inhibitor of oligomeric aggregate formation in vitro and in vivo. Therefore, anle138b is considered a promising drug candidate to beneficially interfere with neurodegenerative processes causing devastating pathologies in humans. The atomistic details of the aggregation inhibition mechanism, however, are to date unknown since the ensemble of small nonfibrillar aggregates is structurally heterogeneous and inaccessible to direct structural characterization. Here, we set out to elucidate anle138b's mode of action using all-atom molecular dynamics simulations on the multi-microsecond time scale. By comparing simulations of dimeric to tetrameric aggregates from fragments of four amyloidogenic proteins (Aß, hTau40, hIAPP, and Sup35N) in the presence and absence of anle138b, we show that the compound reduces the overall number of intermolecular hydrogen bonds, disfavors the sampling of the aggregated state, and remodels the conformational distributions within the small oligomeric peptide aggregates. Most notably, anle138b preferentially interacts with the disordered structure ensemble via its pyrazole moiety, thereby effectively blocking interpeptide main chain interactions and impeding the spontaneous formation of ordered ß-sheet structures, in particular those with out-of-register antiparallel ß-strands. The structurally very similar compound anle234b was previously identified as inactive by in vitro experiments. Here, we show that anle234b has no significant effect on the aggregation process in terms of reducing the ß-structure content. Moreover, we demonstrate that the hydrogen bonding capabilities are autoinhibited due to steric effects imposed by the molecular geometry of anle234b and thereby indirectly confirm the proposed inhibitory mechanism of anle138b. We anticipate that the prominent binding of anle138b to partially disordered and dynamical aggregate structures is a generic basis for anle138b's ability to suppress toxic oligomer formation in a wide range of amyloidogenic peptides and proteins.

An Atomistic View of Amyloidogenic Self-assembly: Structure and Dynamics of Heterogeneous Conformational States in the Pre-nucleation Phase.

The formation of well-defined filamentous amyloid structures involves a polydisperse collection of oligomeric states for which relatively little is known in terms of structural organization. Here we use extensive, unbiased explicit solvent molecular dynamics (MD) simulations to investigate the structural and dynamical features of oligomeric aggregates formed by a number of highly amyloidogenic peptides at atomistic resolution on the µs time scale. A consensus approach has been adopted to analyse the simulations in multiple force fields, yielding an in-depth characterization of pre-fibrillar oligomers and their global and local structure properties. A collision cross section analysis revealed structurally heterogeneous aggregate ensembles for the individual oligomeric states that lack a single defined quaternary structure during the pre-nucleation phase. To gain insight into the conformational space sampled in early aggregates, we probed their substructure and found emerging ß-sheet subunit layers and a multitude of ordered intermolecular ß-structure motifs with growing aggregate size. Among those, anti-parallel out-of-register ß-strands compatible with toxic ß-barrel oligomers were particularly prevalent already in smaller aggregates and formed prior to ordered fibrillar structure elements. Notably, also distinct fibril-like conformations emerged in the oligomeric state and underscore the notion that pre-nucleated oligomers serve as a critical intermediate step on-pathway to fibrils.

Spontaneous aggregation of the insulin-derived steric zipper peptide VEALYL results in different aggregation forms with common features.

Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-ß spines, so-called steric zippers, suggesting common underlying structural features and aggregation mechanisms. Understanding these mechanisms is a prerequisite for designing fibril-binding compounds and inhibitors of fibril formation. The hexapeptide VEALYL, corresponding to the residues B12-17 of full-length insulin, has been identified as one of these short segments. Here, we analyzed the structures of multiple, morphologically different (fibrillar, microcrystal-like, oligomeric) [(13)C,(15)N]VEALYL samples by solid-state nuclear magnetic resonance complemented with results from molecular dynamics simulations. By performing NHHC/CHHC experiments, we could determine that the ß-strands within a given sheet of the amyloid-like fibrils formed by the insulin hexapeptide VEALYL are stacked in an antiparallel manner, whereas the sheet-to-sheet packing arrangement was found to be parallel. Experimentally observed secondary chemical shifts for all aggregate forms, as well as Ø and ψ backbone torsion angles calculated with TALOS, are indicative of ß-strand conformation, consistent with the published crystal structure (PDB ID: 2OMQ). Thus, we could demonstrate that the structural features of all the observed VEALYL aggregates are in agreement with the previously observed homosteric zipper spine packing in the crystalline state, suggesting that several distinct aggregate morphologies share the same molecular architecture.

Driving forces and structural determinants of steric zipper peptide oligomer formation elucidated by atomistic simulations.

Understanding the structural and energetic requirements of non-fibrillar oligomer formation harbors the potential to decipher an important yet still elusive part of amyloidogenic peptide and protein aggregation. Low-molecular-weight oligomers are described to be transient and polymorphic intermediates in the nucleated self-assembly process to highly ordered amyloid fibers and were additionally found to exhibit a profound cytotoxicity. However, detailed structural information on the oligomeric species involved in the nucleation cannot be readily inferred from experiments. Here, we study the spontaneous assembly of steric zipper peptides from the tau protein, insulin and α-synuclein with atomistic molecular dynamics simulations on the microsecond timescale. Detailed analysis of the forces driving the oligomerization reveals a common two-step process akin to a general condensation-ordering mechanism and thus provides a rational understanding of the molecular basis of peptide self-assembly. Our results suggest that the initial formation of partially ordered peptide oligomers is governed by the solvation free energy, whereas the dynamical ordering and emergence of ß-sheets are mainly driven by optimized inter-peptide interactions in the collapsed state. A novel mapping technique based on collective coordinates is employed to highlight similarities and differences in the conformational ensemble of small oligomer structures. Elucidating the dynamical and polymorphic ß-sheet oligomer conformations at atomistic detail furthermore suggests complementary sheet packing characteristics similar to steric zipper structures, but with a larger heterogeneity in the strand alignment pattern and sheet-to-sheet arrangements compared to the cross-ß motif found in the fibrillar or crystalline states.

Mapping the conformational dynamics and pathways of spontaneous steric zipper Peptide oligomerization.

The process of protein misfolding and self-assembly into various, polymorphic aggregates is associated with a number of important neurodegenerative diseases. Only recently, crystal structures of several short peptides have provided detailed structural insights into -sheet rich aggregates, known as amyloid fibrils. Knowledge about early events of the formation and interconversion of small oligomeric states, an inevitable step in the cascade of peptide self-assembly, however, remains still limited. We employ molecular dynamics simulations in explicit solvent to study the spontaneous aggregation process of steric zipper peptide segments from the tau protein and insulin in atomistic detail. Starting from separated chains with random conformations, we find a rapid formation of structurally heterogeneous, -sheet rich oligomers, emerging from multiple bimolecular association steps and diverse assembly pathways. Furthermore, our study provides evidence that aggregate intermediates as small as dimers can be kinetically trapped and thus affect the structural evolution of larger oligomers. Alternative aggregate structures are found for both peptide sequences in the different independent simulations, some of which feature characteristics of the known steric zipper conformation (e.g., -sheet bilayers with a dry interface). The final aggregates interconvert with topologically distinct oligomeric states exclusively via internal rearrangements. The peptide oligomerization was analyzed through the perspective of a minimal oligomer, i.e., the dimer. Thereby all observed multimeric aggregates can be consistently mapped onto a space of reduced dimensionality. This novel method of conformational mapping reveals heterogeneous association and reorganization dynamics that are governed by the characteristics of peptide sequence and oligomer size.