Pyroglutamate-modified amyloid ß(3-42) monomer has more ß-sheet content than the amyloid ß(1-42) monomer.

The aggregation of the amyloid ß (Aß) peptide is a major hallmark of Alzheimer's disease. This peptide can aggregate into oligomers, proto-fibrils and mature fibrils, which eventually assemble into amyloid plaques in vivo. Several post-translational modifications lead to the presence of different forms of the Aß peptide in the amyloid plaques with different biophysical and biochemical properties. While the canonical forms Aß(1-40) and Aß(1-42) have been found to be the major components of amyloid plaques, N-terminally pyroglutamate-modified variants, specifically pE-Aß(3-42), amount to a significant fraction of the total Aß plaque content of AD brains. With increased hydrophobicity, these variants display a more pronounced aggregation behaviour in vitro which, together with their higher stability against degradation in vivo is thought to make them crucial molecular players in the aetiology of AD. The peptide monomers are the smallest assembly units, and play an important role in most of the individual molecular processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. Understanding the monomeric conformational ensembles of the isoforms is important in unraveling observed differences in their bio-physico-chemical properties. Here we use enhanced and extensive molecular dynamics simulations to study the structural flexibility of the N-terminally truncated Pyroglutamate modified isomer of Aß, pE-Aß(3-42) monomer, and compared it with simulations of the Aß(1-42) peptide monomer under the same conditions. We find significant differences, especially in the secondary structure and hydrophobic exposure, which might be responsible for their different behaviour in biophysical experiments.

Large-scale, dynamin-like motions of the human guanylate binding protein 1 revealed by multi-resolution simulations.

Guanylate binding proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible protein that undergoes a hinge motion similar to the movements observed for other dynamin-like proteins. Another large-scale motion was observed for the C-terminal helix α13, providing a molecular view for the α13-α13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, revealing why GTP binding is needed for hGBP1 dimerization to occur.

Bioactivation of Quinolines in a Recombinant Estrogen Receptor Transactivation Assay Is Catalyzed by N-Methyltransferases.

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17ß-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Pathways of Amyloid-ß Aggregation Depend on Oligomer Shape.

One of the main research topics related to Alzheimer's disease is the aggregation of the amyloid-ß peptide, which was shown to follow different pathways for the two major alloforms of the peptide, Aß40 and the more toxic Aß42. Experimental studies emphasized that oligomers of specific sizes appear in the early aggregation process in different quantities and might be the key toxic agents for each of the two alloforms. We use transition networks derived from all-atom molecular dynamics simulations to show that the oligomers leading to the type of oligomer distributions observed in experiments originate from compact conformations. Extended oligomers, on the other hand, contribute more to the production of larger aggregates thus driving the aggregation process. We further demonstrate that differences in the aggregation pathways of the two Aß alloforms occur as early as during the dimer stage. The higher solvent-exposure of hydrophobic residues in Aß42 oligomers contributes to the different aggregation pathways of both alloforms and also to the increased cytotoxicity of Aß42.

Understanding Amyloid-ß Oligomerization at the Molecular Level: The Role of the Fibril Surface.

The aggregation of the amyloid ß-peptide into fibrils is a complex process that involves mechanisms such as primary and secondary nucleation, fibril elongation and fibril fragmentation. Some of these processes generate neurotoxic Aß oligomers, which are involved in the development of Alzheimer's disease. Recent experimental studies have emphasized the role of the fibril as a catalytic surface for the production of highly toxic oligomers during secondary nucleation. By using molecular dynamics simulations, we show that it is the hydrophobic fibril region that causes the structural changes required for catalyzing the formation of ß-sheet-rich Aß1-42 oligomers on the fibril surface. These results reveal, for the first time, the molecular basis of the secondary nucleation pathway.

Selective refinement and selection of near-native models in protein structure prediction.

In recent years in silico protein structure prediction reached a level where fully automated servers can generate large pools of near-native structures. However, the identification and further refinement of the best structures from the pool of models remain problematic. To address these issues, we have developed (i) a target-specific selective refinement (SR) protocol; and (ii) molecular dynamics (MD) simulation based ranking (SMDR) method. In SR the all-atom refinement of structures is accomplished via the Rosetta Relax protocol, subject to specific constraints determined by the size and complexity of the target. The best-refined models are selected with SMDR by testing their relative stability against gradual heating through all-atom MD simulations. Through extensive testing we have found that Mufold-MD, our fully automated protein structure prediction server updated with the SR and SMDR modules consistently outperformed its previous versions.

Folding of pig gastric mucin non-glycosylated domains: a discrete molecular dynamics study.

Mucin glycoproteins consist of tandem-repeating glycosylated regions flanked by non-repetitive protein domains with little glycosylation. These non-repetitive domains are involved in polymerization of mucin and play an important role in the pH-dependent gelation of gastric mucin, which is essential for protecting the stomach from autodigestion. We examine folding of the non-repetitive sequence of PGM-2X (242 amino acids) and the von Willebrand factor vWF-C1 domain (67 amino acids) at neutral and low pH using discrete molecular dynamics (DMD) in an implicit solvent combined with a four-bead peptide model. Using the same implicit solvent parameters, folding of both domains is simulated at neutral and low pH. In contrast to vWF-C1, PGM-2X folding is strongly affected by pH as indicated by changes in the contact order, radius of gyration, free-energy landscape, and the secondary structure. Whereas the free-energy landscape of vWF-C1 shows a single minimum at both neutral and low pH, the free-energy landscape of PGM-2X is characterized by multiple minima that are more numerous and shallower at low pH. Detailed structural analysis shows that PGM-2X partially unfolds at low pH. This partial unfolding is facilitated by the C-terminal region GLU236-PRO242, which loses contact with the rest of the domain due to effective "mean-field" repulsion among highly positively charged N- and C-terminal regions. Consequently, at low pH, hydrophobic amino acids are more exposed to the solvent. In vWF-C1, low pH induces some structural changes, including an increased exposure of CYS at position 67, but these changes are small compared to those found in PGM-2X. For PGM-2X, the DMD-derived average ß-strand propensity increases from 0.26 ± 0.01 at neutral pH to 0.38 ± 0.01 at low pH. For vWF-C1, the DMD-derived average ß-strand propensity is 0.32 ± 0.02 at neutral pH and 0.35 ± 0.02 at low pH. The DMD-derived structural information provides insight into pH-induced changes in the folding of two distinct mucin domains and suggests plausible mechanisms of the aggregation/gelation of mucin.

Interaction of Therapeutic d-Peptides with Aß42 Monomers, Thermodynamics, and Binding Analysis.

The aggregation of the amyloid-ß (Aß) peptide is a major hallmark of Alzheimer's disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. Several d-peptides have been confirmed as promising candidates to inhibit the aggregation of Aß into toxic oligomers and fibrils by specifically interacting with monomeric species. In this work, we elucidate the structural interaction and thermodynamics of binding between three d-peptides (D3, ANK6, and RD2) and Aß42 monomers by means of enhanced molecular dynamics simulations. Our study derives thermodynamic energies in good agreement with experimental values and suggests that there is an enhanced binding for D3 and ANK6, which leads to more stable complexes than for RD2. The binding of D3 to Aß42 is shown to be weakly exothermic and mainly entropically driven, whereas the complex formation between the ANK6 and RD2 with the Aß42 free monomer is weakly endothermic. In addition, the changes in the solvent-accessible surface area and the radius of gyration support that the binding between Aß42 and d-peptides is mainly driven by electrostatic and hydrophobic interactions and leads to more compact conformations.

Molecular Dynamics Simulations of Protein Aggregation: Protocols for Simulation Setup and Analysis with Markov State Models and Transition Networks.

Protein disorder and aggregation play significant roles in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. The end products of the aggregation process in these diseases are highly structured amyloid fibrils. Though in most cases, small, soluble oligomers formed during amyloid aggregation are the toxic species. A full understanding of the physicochemical forces that drive protein aggregation is thus required if one aims for the rational design of drugs targeting the formation of amyloid oligomers. Among a multitude of biophysical and biochemical techniques that are employed for studying protein aggregation, molecular dynamics (MD) simulations at the atomic level provide the highest temporal and spatial resolution of this process, capturing key steps during the formation of amyloid oligomers. Here we provide a step-by-step guide for setting up, running, and analyzing MD simulations of aggregating peptides using GROMACS. For the analysis, we provide the scripts that were developed in our lab, which allow to determine the oligomer size and inter-peptide contacts that drive the aggregation process. Moreover, we explain and provide the tools to derive Markov state models and transition networks from MD data of peptide aggregation.

Compact fibril-like structure of amyloid ß-peptide (1-42) monomers.

Amyloid ß (Aß) monomers are the smallest assembly units, and play an important role in most of the individual processes involved in amyloid fibril formation. An important question is whether the monomer can adopt transient fibril-like conformations in solution. Here we use enhanced sampling simulations to study the Aß42 monomer structural flexibility. We show that the monomer frequently adopts conformations with the N-terminus region structured very similarly to the conformation it adopts inside the fibril. This intrinsic propensity of monomeric Aß to adopt fibril-like conformations could explain the low free energy barrier for Aß42 fibril elongation.

MUFOLD: A new solution for protein 3D structure prediction.

There have been steady improvements in protein structure prediction during the past 2 decades. However, current methods are still far from consistently predicting structural models accurately with computing power accessible to common users. Toward achieving more accurate and efficient structure prediction, we developed a number of novel methods and integrated them into a software package, MUFOLD. First, a systematic protocol was developed to identify useful templates and fragments from Protein Data Bank for a given target protein. Then, an efficient process was applied for iterative coarse-grain model generation and evaluation at the Calpha or backbone level. In this process, we construct models using interresidue spatial restraints derived from alignments by multidimensional scaling, evaluate and select models through clustering and static scoring functions, and iteratively improve the selected models by integrating spatial restraints and previous models. Finally, the full-atom models were evaluated using molecular dynamics simulations based on structural changes under simulated heating. We have continuously improved the performance of MUFOLD by using a benchmark of 200 proteins from the Astral database, where no template with >25% sequence identity to any target protein is included. The average root-mean-square deviation of the best models from the native structures is 4.28 A, which shows significant and systematic improvement over our previous methods. The computing time of MUFOLD is much shorter than many other tools, such as Rosetta. MUFOLD demonstrated some success in the 2008 community-wide experiment for protein structure prediction CASP8.

Membrane curvature and surface area per lipid affect the conformation and oligomeric state of HIV-1 fusion peptide: a combined FTIR and MD simulation study.

Fourier-transformed infrared spectroscopy (FTIR) and molecular dynamics (MD) simulation results are presented to support our hypothesis that the conformation and the oligomeric state of the HIV-1 gp41 fusion domain or fusion peptide (gp41-FP) are determined by the membrane surface area per lipid (APL), which is affected by the membrane curvature. FTIR of the gp41-FP in the Aerosol-OT (AOT) reversed micellar system showed that as APL decreases from approximately 50 to 35 A2 by varying the AOT/water ratio, the FP changes from the monomeric alpha-helical to the oligomeric beta-sheet structure. MD simulations in POPE lipid bilayer systems showed that as the APL decreases by applying a negative surface tension, helical monomers start to unfold into turn-like structures. Furthermore, an increase in the applied lateral pressure during nonequilibrium MD simulations favored the formation of beta-sheet structure. These results provide better insight into the relationship between the structures of the gp41-FP and the membrane, which is essential in understanding the membrane fusion process. The implication of the results of this work on what is the fusogenic structure of the HIV-1 FP is discussed.

Aß under stress: the effects of acidosis, Cu2+-binding, and oxidation on amyloid ß-peptide dimers.

In light of the high affinity of Cu2+ for Alzheimer's Aß1-42 and its ability to subsequently catalyze the formation of radicals, we examine the effects of Cu2+ binding, Aß oxidation, and an acidic environment on the conformational dynamics of the smallest Aß1-42 oligomer, the Aß1-42 dimer. Transition networks calculated from Hamiltonian replica exchange molecular dynamics (H-REMD) simulations reveal that the decreased pH considerably increased the ß-sheet content, whereas Cu2+ binding increased the exposed hydrophobic surface area, both of which can contribute to an increased oligomerization propensity and toxicity.

Fully Atomistic Aß40 and Aß42 Oligomers in Water: Observation of Porelike Conformations.

Oligomers formed by amyloid ß-protein (Aß) are central to Alzheimer's disease (AD) pathology, yet their structure remains elusive. Of the two predominant Aß alloforms, Aß40 and Aß42, the latter is more strongly associated with AD. Here, we structurally characterized Aß40 and Aß42 monomers through pentamers which were converted from previously derived coarse-grained (DMD4B-HYDRA) simulations into all-atom conformations and subjected to explicit-solvent MD. Free energy landscapes revealed that structural differences between Aß40 and Aß42 conformations increase with oligomer order up to trimers. All conformations display high statistical coil and turn content (40-50%) with minor ß-strand and α-helical content (<10%). Aß40 tetramers and pentamers exhibit significantly more elongated morphologies than the respective Aß42 conformations. Unlike the initial DMD4B-HYDRA conformations, fully atomistic Aß40 and Aß42 trimers, tetramers, and pentamers form water-permeable pores, whereby the tendency for pore formation sharply increased with oligomer order and is the highest for Aß42 pentamers. Previous studies reported that Aß oligomers form ion channels when embedded into a cellular membrane, which causes an abnormal ion flux and eventually leads to cell death. Our findings reveal an extraordinary ability of Aß oligomers to form pores in pure water prior to their insertion into a membrane and thus provide support to the ion channel hypothesis of AD.

Aß42 pentamers/hexamers are the smallest detectable oligomers in solution.

Amyloid ß (Aß) oligomers may play a decisive role in Alzheimer's disease related neurodegeneration, but their structural properties are poorly understood. In this report, sedimentation velocity centrifugation, small angle neutron scattering (SANS) and molecular modelling were used to identify the small oligomeric species formed by the 42 amino acid residue long isoform of Aß (Aß42) in solution, characterized by a sedimentation coefficient of 2.56 S, and a radius of gyration between 2 and 4 nm. The measured sedimentation coefficient is in close agreement with the sedimentation coefficient calculated for Aß42 hexamers using MD simulations at µM concentration. To the best of our knowledge this is the first report detailing the Aß42 oligomeric species by SANS measurements. Our results demonstrate that the smallest detectable species in solution are penta- to hexamers. No evidences for the presence of dimers, trimers or tetramers were found, although the existence of those Aß42 oligomers at measurable quantities had been reported frequently.

Minimal model of self-assembly: emergence of diversity and complexity.

Molecular self-assembly is ubiquitous in nature, yet prediction of assembly pathways from fundamental interparticle interactions has yet to be achieved. Here, we introduce a minimal self-assembly model with two attractive and two repulsive beads bound into a tetrahedron. The model is associated with a single parameter η defined as the repulsive to attractive interaction ratio. We explore self-assembly pathways and resulting assembly morphologies for different η values by discrete molecular dynamics. Our results demonstrate that η governs the assembly dynamics and resulting assembly morphologies, revealing an unexpected diversity and complexity for 0.5 ≤ η < 1. One of the key processes that governs the assembly dynamics is assembly breakage, which emerges spontaneously at η > 0 with the breakage rate increasing with η. The observed assembly pathways display a broad variety of assembly structures characteristic of aggregation of amyloidogenic proteins, including quasi-spherical oligomers that coassemble into elongated protofibrils, followed by a conversion into ordered polymorphic fibril-like aggregates. We further demonstrate that η can be meaningfully mapped onto amyloidogenic protein sequences, with the majority of amyloidogenic proteins characterized by 0.5 ≤ η < 1. Prion proteins, which are known to form highly breakage-prone fibrils, are characterized by η > 1, consistent with the model predictions. Our model thus provides a theoretical basis for understanding the universal aspects of aggregation pathways of amyloidogenic proteins relevant to human disease. As the model is not specific to proteins, these findings represent an important step toward understanding and predicting assembly dynamics of not only proteins but also viruses, colloids, and nanoparticles.

Early amyloid ß-protein aggregation precedes conformational change.

The aggregation of amyloid-ß protein (1-42) is studied at experimental concentrations using all-atom molecular dynamics simulations. We observe a fast aggregation into oligomers without significant changes in the internal structure of individual proteins. The aggregation process is characterized in terms of transition networks.

A kinetic approach to the sequence-aggregation relationship in disease-related protein assembly.

It is generally accepted that oligomers of aggregating proteins play an important role in the onset of neurodegenerative diseases. While in silico aggregation studies of full length amyloidogenic proteins are computationally expensive, the assembly of short protein fragments derived from these proteins with similar aggregating properties has been extensively studied. In the present work, molecular dynamics simulations are performed to follow peptide aggregation on the microsecond time scale. By defining aggregation states, we identify transition networks, disconnectivity graphs, and first passage time distributions to describe the kinetics of the assembly process. This approach unravels differences in the aggregation into hexamers of two peptides with different primary structures. The first is GNNQQNY, a hydrophilic fragment from the prion protein Sup35, and the second is KLVFFAE, a fragment from amyloid-ß protein, with a hydrophobic core delimited by two charged amino acids. The assembly of GNNQQNY suggests a mechanism of monomer addition, with a bias toward parallel peptide pairs and a gradual increase in the amount of ß-strand content. For KLVFFAE, a mechanism involving dimers rather than monomers is revealed, involving a generally higher ß-strand content and a transition toward a larger number of antiparallel peptide pairs during the rearrangement of the hexamer. The differences observed for the aggregation of the two peptides suggests the existence of a sequence-aggregation relationship.

Dimer formation enhances structural differences between amyloid ß-protein (1-40) and (1-42): an explicit-solvent molecular dynamics study.

Amyloid ß-protein (Aß) is central to the pathology of Alzheimer's disease. A 5% difference in the primary structure of the two predominant alloforms, Aß(1-40) and Aß(1-42), results in distinct assembly pathways and toxicity properties. Discrete molecular dynamics (DMD) studies of Aß(1-40) and Aß(1-42) assembly resulted in alloform-specific oligomer size distributions consistent with experimental findings. Here, a large ensemble of DMD-derived Aß(1-40) and Aß(1-42) monomers and dimers was subjected to fully atomistic molecular dynamics (MD) simulations using the OPLS-AA force field combined with two water models, SPCE and TIP3P. The resulting all-atom conformations were slightly larger, less compact, had similar turn and lower ß-strand propensities than those predicted by DMD. Fully atomistic Aß(1-40) and Aß(1-42) monomers populated qualitatively similar free energy landscapes. In contrast, the free energy landscape of Aß(1-42) dimers indicated a larger conformational variability in comparison to that of Aß(1-40) dimers. Aß(1-42) dimers were characterized by an increased flexibility in the N-terminal region D1-R5 and a larger solvent exposure of charged amino acids relative to Aß(1-40) dimers. Of the three positively charged amino acids, R5 was the most and K16 the least involved in salt bridge formation. This result was independent of the water model, alloform, and assembly state. Overall, salt bridge propensities increased upon dimer formation. An exception was the salt bridge propensity of K28, which decreased upon formation of Aß(1-42) dimers and was significantly lower than in Aß(1-40) dimers. The potential relevance of the three positively charged amino acids in mediating the Aß oligomer toxicity is discussed in the light of available experimental data.