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Assessing Polyglutamine Conformation in the Nucleating Event by Molecular Dynamics Simulations
Abstract
Polyglutamine (polyQ) diseases comprise a group of dominantly inherited pathology caused by an expansion of an unstable polyQ stretch which is presumed to form β-sheets. Similar to other amyloid pathologies, polyQ amyloidogenesis occurs via a nucleated polymerization mechanism, and proceeds through energetically unfavorable nucleus whose existence and structure are difficult to detect. Here, we use atomistic molecular dynamics simulations in explicit solvent to assess the conformation of the polyQ stretch in the nucleus that initiates polyQ fibrillization. Comparison of the kinetic stability of various structures of polyQ peptide with a Q-length in the pathological range (Q(40)) revealed that steric zipper or nanotube-like structures (β-nanotube or β-pseudohelix) are not kinetically stable enough to serve as a template to initiate polyQ fibrillization as opposed to β-hairpin-based (β-sheet and β-sheetstack) or α-helical conformations. The selection of different structures of the polyQ stretch in the aggregation-initiating event may provide an alternative explanation for polyQ aggregate polymorphism.
... All peptides consisted of 40 glutamine residues (Q 40 ), with NH 2 /COOH termini. Their stability as monomers has been studied previously (47). ...
... When using the docking approach, one makes the implicit assumption that no major monomer restructuring occurs upon dimerization; here, this assumption appears plausible, as the highest free energy state is known to be a structured monomer (Fig. 1). The initial monomer structures for the conformers were the same as in our previous study (47). The docking scores acquired from the calculations provided qualitative information on the stability of dimers (under the assumption of static conformers) and allowed for a rough comparison of their elongation potentials. ...
... Dimers of Q 40 were built by docking monomeric structures proposed in the literature. We had previously explored their kinetic stabilities as monomers and shown that both a-helical and b-hairpin-containing monomers have average lifetimes exceeding 100 ns (47). With the exception of the a-helix, all the conformers gained kinetic stability upon dimerization. ...
A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β-hairpin-containing conformers act as templates for fibril formation.
... It is nontrivial to structurally characterize the Httexon-1 protein due to its inherently disordered nature. It is also known that when the other two domains of Htt exon-1 protein, N17 (the first 17 residues of the Htt N-terminal region) and C38 (38 residues near the Httexon-1 C-terminus, polyproline-rich segment), are absent and the Q-length is 20 or greater, polyQ collapses into a disordered but compact globule (Perutz et al., 1994;Chen et al., 2002;Crick et al., 2006;Miettinen et al., 2012;Heck et al., 2014;Hoop et al., 2016). The supercompact structures of polyQ (not found in other globular proteins) are mainly owing to the "glue-like" propensity of glutamine sidechains to form many hydrogen bonds (Kang et al., 2017). ...
... PolyQ peptides are known to form hydrogen bonds using both the glutamine backbone and sidechain which is the main cause of the formation of polyQ supercompact structures (Perutz et al., 1994;Chen et al., 2002;Crick et al., 2006;Miettinen et al., 2012;Heck et al., 2014;Hoop et al., 2016;Kang et al., 2017). Here, we analyze the time evolution of polyQ hydrogen bonds and find interesting competition behavior between the intra-polyQ interactions (mainly hydrogen bonds) and polyQ sidechain-nanosheet interactions. ...
There is a strong negative correlation between the polyglutamine (polyQ) domain length (Q-length) in the intrinsically disordered Huntingtin protein (Htt) exon-1 and the age of onset of Huntington's disease (HD). PolyQ of Q-length longer than 40 has the propensity of forming very compact aggregate structures, leading to HD at full penetrance. Recent advances in nanobiotechnology provided a new platform for the development of novel diagnosis and therapeutics. Here, we explore the possibility of utilizing 2D-nanomaterials to inhibit the formation of supercompact polyQ structures through the so-called “folding-upon-binding” where the protein structure is dependent on the binding substrate. Using molecular dynamics simulations, we characterize two polyQ peptides with Q-length of 22 (Q22, normal length) and 46 (Q46, typical length causing HD) binding to both graphene and molybdenum disulfide (MoS2) nanosheets, which have been applied as antibacterial or anticancer agents. Upon binding, Q22 unfolds and elongates on both grapheme and MoS2 surfaces, regardless of its initial conformation, with graphene showing slightly stronger effect. In contrast, initially collapsed Q46 remains mostly collapsed within our simulation time on both nanosheets even though they do provide some “stretching” to Q46 as well. Further analyses indicate that the hydrophobic nature of graphene/MoS2 promotes the stretching of polyQ on nanosheets. However, there is strong competition with the intra-polyQ interactions (mainly internal hydrogen bonds) leading to the disparate folding/binding behaviors of Q22 and Q46. Our results present distinct Q-length specific behavior of the polyQ domain upon binding to two types of 2D-nanomaterials which holds clinical relevance for Huntington's disease.
... (e) Schematic free energy profile of fibril formation and growth, for short and long polyglutamine, based on published nucleation and fiber elongation energy values. 96,97,99,100 4 Experimental Biology and Medicine by an independent X-ray study, which reported subtle variations among different-length polyglutamine peptides and proposed distinct structures with kinked side chains. 55 Thus, while X-ray studies provided unambiguous evidence of an amyloid-like architecture that was shared by aggregated polyglutamine and HttEx1, they were unable to resolve a unique atomic structure. ...
... The rate-limiting step for long polyglutamine manifests instead as a monomeric event. 100,101 Various lines of evidence support the idea that monomeric nucleation reflects the formation of a b-hairpin within the expanded polyglutamine monomer. Structural studies detect b-hairpin structures in the endproduct of aggregated polyglutamine and Q44-HttEx1. ...
Huntington’s disease, like other neurodegenerative diseases, continues to lack an effective cure. Current treatments that address early symptoms ultimately fail Huntington’s disease patients and their families, with the disease typically being fatal within 10–15 years from onset. Huntington’s disease is an inherited disorder with motor and mental impairment, and is associated with the genetic expansion of a CAG codon repeat encoding a polyglutamine-segment-containing protein called huntingtin. These Huntington’s disease mutations cause misfolding and aggregation of fragments of the mutant huntingtin protein, thereby likely contributing to disease toxicity through a combination of gain-of-toxic-function for the misfolded aggregates and a loss of function from sequestration of huntingtin and other proteins. As with other amyloid diseases, the mutant protein forms non-native fibrillar structures, which in Huntington’s disease are found within patients’ neurons. The intracellular deposits are associated with dysregulation of vital processes, and inter-neuronal transport of aggregates may contribute to disease progression. However, a molecular understanding of these aggregates and their detrimental effects has been frustrated by insufficient structural data on the misfolded protein state. In this review, we examine recent developments in the structural biology of polyglutamine-expanded huntingtin fragments, and especially the contributions enabled by advances in solid-state nuclear magnetic resonance spectroscopy. We summarize and discuss our current structural understanding of the huntingtin deposits and how this information furthers our understanding of the misfolding mechanism and disease toxicity mechanisms.
Impact statement
Many incurable neurodegenerative disorders are associated with, and potentially caused by, the amyloidogenic misfolding and aggregation of proteins. Usually, complex genetic and behavioral factors dictate disease risk and age of onset. Due to its principally mono-genic origin, which strongly predicts the age-of-onset by the extent of CAG repeat expansion, Huntington’s disease (HD) presents a unique opportunity to dissect the underlying disease-causing processes in molecular detail. Yet, until recently, the mutant huntingtin protein with its expanded polyglutamine domain has resisted structural study at the atomic level. We present here a review of recent developments in HD structural biology, facilitated by breakthrough data from solid-state NMR spectroscopy, electron microscopy, and complementary methods. The misfolded structures of the fibrillar proteins inform our mechanistic understanding of the disease-causing molecular processes in HD, other CAG repeat expansion disorders, and, more generally, protein deposition disease.
... These structures are the only crystal structures of poly-Q segments available in the RCSB PDB database. Computational modeling can provide valuable insights to this problem [23,30,31], but to our knowledge no comprehensive studies have been reported comparing the 3D structures predicted for these segments with the limited experimental data available. ...
... None-the-less, both programs produce a reasonable ensemble of structures with sufficient diversity and without extreme deviations. Several studies have illustrated that the poly-Q repeat regions of these proteins are highly disordered with structure flexibility [31], but this has not been quantified experimentally. Therefore it is challenging to discriminate among these two approaches using these criteria. ...
Background
The expansion of polyglutamine (poly-Q) repeats in several unrelated proteins is associated with at least ten neurodegenerative diseases. The length of the poly-Q regions plays an important role in the progression of the diseases. The number of glutamines (Q) is inversely related to the onset age of these polyglutamine diseases, and the expansion of poly-Q repeats has been associated with protein misfolding. However, very little is known about the structural changes induced by the expansion of the repeats. Computational methods can provide an alternative to determine the structure of these poly-Q proteins, but it is important to evaluate their performance before large scale prediction work is done.
Results
In this paper, two popular protein structure prediction programs, I-TASSER and Rosetta, have been used to predict the structure of the N-terminal fragment of a protein associated with Huntington's disease with 17 glutamines. Results show that both programs have the ability to find the native structures, but I-TASSER performs better for the overall task.
Conclusions
Both I-TASSER and Rosetta can be used for structure prediction of proteins with poly-Q repeats. Knowledge of poly-Q structure may significantly contribute to development of therapeutic strategies for poly-Q diseases.
... Two types of aa repetitions can be distinguished: (i) aa-rich segments/regions of dense diffuse unique aa occurrence and (ii) segments with monotonous aa sequences including groups of frequent hydrophobic zippers and hydrophilic or charged poly-aa regions composing beta-hairpin based or alpha-helical conformations and sometimes possibly also polar zippers or nanotube-like structures (cf. Ref. [120]). Since aa repetitions are less described in reviews, we further deal with them in more detail. ...
... The pathogenic mechanism includes protein conformation changes possibly accompanied by selection of PolyQ structures. Such changes result in the formation of widely known beta sheet-rich aggregates [120,130]. ...
This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamical properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamical changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships.
... The other configuration has the first and fourth strands paired and flanking one side of the central hairpin (Fig. S8B). This hypothetical fold of polyQ, along with the alternative configuration having two hairpins and one arch, had previously been simulated by multiple groups and found to be more stable than any single sheet or parallel stranded zipper configuration (Man et al., 2015;Miettinen et al., 2012). We 11 . ...
A long-standing goal of the study of amyloids has been to characterize the physical nature of the rate-determining nucleating event. However, the transience and rarity of that event within the heterogeneous ensemble of states populated by amyloid-forming proteins make it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we address these limitations by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve independent nucleation events. We characterized over one hundred rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. We deduce that amyloid formation by polyQ begins with a steric zipper embryo of approximately twelve interdigitated glutamine side chains within an individual polypeptide molecule. Formation of the embryo was limited to polypeptides longer than the pathogenic threshold, and involved neither phase separation nor oligomerization. We found that different amyloid propensities of polyQ sequence variants can be rationalized by steric zipper ordering orthogonally to the axis of polymerization, and validated this intuition using all-atom molecular dynamics simulations. The unique ability of the polyQ sequence to fold in this fashion not only allowed for polyQ amyloid to nucleate from low concentrations; it also stalled amyloid growth with a concomitant accumulation of partially-ordered oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.
... 112 The structural stability and the aggregation properties were extensively discussed in the literature. 65,114,115 The conformational landscape presented in the three studies, structures turning into steric zippers and β-helix, was not in agreement with the conformational states found by Gomez-Sicilia et al., 112 because they considered monomers and not dimers. ...
Over the recent years, Huntington’s disease (HD) has become widely discussed in the scientific literature especially because, at the mutant level, there are several contradictions regarding the aggregation mechanism. The specific role of the physiological huntingtin protein remains unknown, due to the lack of characterization of its entire crystallographic structure, making the experimental and theoretical research even harder when taking into consideration its involvement into multiple biological functions and its high affinity for different interacting partners. Different types of models, containing fewer (not more than 35 Qs) polyglutamine residues for the WT structure and above 35 Qs for the mutants, were subjected to classical or advanced MD simulations to establish the proteins’ structural stability by evaluating their conformational changes. Outside the polyQ tract, there are two other regions of interest (the N17 domain and the polyP rich domain) considered to be essential for the aggregation kinetics at the mutant level. The polymerization process is considered to be dependent on the polyQ length. As the polyQ tract’s dimension increases the structures present more β-sheet conformations. Contrary, it is also considered that the aggregation stability is not necessarily dependent on the number of Qs, while the initial stage of the aggregation seed might play the decisive role. A general assumption regarding the polyP domain is that it might preserve the polyQ structures soluble by acting as an antagonist for β-sheet formation.
... These timescales are 12 orders of magnitude shorter than the timescale for forming any secondary structure. To exclude the formation of new secondary structures, we further performed 300-ns MD simulations for apo-AdcA and Zn2-AdcA, a timescale which is sufficient to represent the conformational change of complex proteins (24,25). Compared with the initial state, there was no obvious change in conformation or hydrogen bonds in the equilibrium state ( Fig. S5A-C). ...
Zinc is an essential metal in bacteria. One important bacterial zinc transporter is AdcA, and most bacteria possess AdcA homologs that are single-domain small proteins owing to better efficiency of protein biogenesis. However, a double-domain AdcA with two zinc-binding sites is significantly overrepresented in Streptococcus species, many of which are major human pathogens. Using molecular simulation and experimental validations of AdcA from Streptococcus pyogenes, we found here that the two AdcA domains sequentially stabilize the structure upon zinc binding, indicating an organization required for both increased zinc affinity and transfer speed. This structural organization appears to endow Streptococcus species with distinct advantages in zinc-depleted environments, which would not be achieved by each single AdcA domain alone. This enhanced zinc transport mechanism sheds light on the significance of the evolution of the AdcA domain fusion, provides new insights into double-domain transporter proteins with two binding sites for the same ion, and indicates a potential target of antimicrobial drugs against pathogenic Streptococcus species.
... Theoretical in-silico studies might be of notable help, providing atomistic and structural insights to understand and guide experiments [29][30][31][32]. ...
Polyglutamine peptides with an abnormal repeat length are the causative agent of at least nine genetic diseases, known as polyglutamine diseases, which include Huntigton’s disease (HD) and some ataxias, in between the others. In the case of HD the disease appears when the polyQ segment has approximately 36 glutamines (Q). It is known that conformational changes occur and that polyQ aggregation is modulated by the number of Q present in the chain. In the present study, the conformations of monomeric polyQ chains of different lengths (from 7 Q to 45 Q) are systematically investigated by performing all-atom molecular dynamics simulations in explicit water at 310 K. All simulations are performed without any structure constraints. For each system trajectories of 300 ns are generated. Analysis of structures, radius of gyration, root-mean-squared deviation, hydrogen bond analysis, clustering, dihedral angles and analysis of the secondary structures of all studied polyQ repeats are presented. This study provides information about the conformational changes of the considered monomeric peptides with increasing the number of Q. The obtained results are compared with available data. This work confirms that a structural change from the polyQ segment containing 24 Q to the one containing 37 residues occurs.
... Computational simulations, using a variety of approaches including Molecular Dynamics (MD), Replica Exchange MD (REMD) and Coarse Grain (CG) have been used to study polyQ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 segment aggregation in several publications [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]. The overall picture that emerges from these studies, which include both monomers and dimers of different polyQ lengths, is that polyQ segments tend to favor β-sheet structures, which are propense to aggregation. ...
Protein misfolding and aggregation is a pathogenic feature shared among at least ten polyglutamine (polyQ) neurodegenerative diseases. While solvent-solution interaction is a key factor driving protein folding and aggregation, the solvation properties of expanded polyQ tracts are not well understood. By using GPU-enabled all-atom molecular dynamics simulations of polyQ monomers in an explicit solvent environment, this study shows that solvent-polyQ interaction propensity decreases as the lengths of polyQ tract increases. This study finds a predominance in long-distance interactions between residues far apart in polyQ sequences with longer polyQ segments, that leads to significant conformational differences. This study also indicates that large loops, comprised of parallel β-structures, appear in long polyQ tracts and present new aggregation building blocks with aggregation driven by long-distance intra-polyQ interactions. Finally, consistent with previous observations using coarse-grain simulations, this study demonstrates that there is a gain in the aggregation propensity with increased polyQ length, and that this gain is correlated with decreasing ability of solvent-polyQ interaction. These results suggest the modulation of solvent-polyQ interactions as a possible therapeutic strategy for treating polyQ diseases.
... The concentration of protein in the eye lens is orders of magnitude higher, requiring the remarkable eye lens protein solubility that is based on a careful balancing of repulsive and attractive protein-protein interactions 57 . Amyloidogenic aggregation at low concentrations tends to involve exposure of hydrophobic sites through misfolding, and proceed via specific nucleation events 38,58 . At very high-protein concentrations, aggregation can result from surface-mediated changes in protein-protein interactions, well before large-scale structural changes trigger the formation of an amyloidogenic state. ...
Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils.
... The concentration of protein in the eye lens is orders of magnitude higher, requiring the remarkable eye lens protein solubility that is based on a careful balancing of repulsive and attractive protein-protein interactions 57 . Amyloidogenic aggregation at low concentrations tends to involve exposure of hydrophobic sites through misfolding, and proceed via specific nucleation events 38,58 . At very high-protein concentrations, aggregation can result from surface-mediated changes in protein-protein interactions, well before large-scale structural changes trigger the formation of an amyloidogenic state. ...
Human gamma-D-crystallin (HGD) is an extremely soluble (>400mg/mL in vivo) and stable protein that under certain conditions succumbs to protein aggregation, which is associated with cataract formation. Cataracts are the leading cause of blindness globally. Both environmental and genetic factors enable cataract formation by disrupting the solubility of HGD. Several chemical modifications are thought to be linked to loss of solubility and precipitation of HGD. The P23T mutant of HGD is genetically linked to three distinct congenital cataracts. It is crucial to develop a mechanistic understanding of how the reduced solubility of P23T HGD is caused by chemical and/or structural changes. We performed structural and chemical analyses on aggregated or aggregation-prone HGD to gain a molecular understanding of the aggregation mechanism, as well as the nature and location of chemical modifications.We find P23T HGD is aggregation prone, but forms qualitatively different aggregates as a function of the environmental conditions. Among other factors, we examined the impact of pH and UV irradiation on aggregation and evaluated the resulting aggregates by Electron Microscopy (EM), X-ray powder diffraction, Mass Spectrometry (MS), and multidimensional magic-angle-spinning (MAS) solid-state Nuclear Magnetic Resonance (NMR). Distinct conformations of HGD were observed in the aggregated state by X-ray powder diffraction and EM, and chemical changes were observed by MS. Through comparison to the NMR chemical shifts of the protein in the soluble and aggregated states, we identified structural changes in a residue and site-specific fashion. Our data reveal that aggregation conditions not only affect the macroscopic appearance of the aggregates, but also have a dramatic impact on the aggregates’ internal atomic structure. Therefore, distinct molecular mechanisms for crystallin aggregation exist.
... The cross-beta structure was confirmed by solid state-NMR on exactly the same D2Q15K2 peptides (Schneider et al. 2011) and both cross-beta and the characteristic steric zipper of the side chains in poly-Q amyloid fibers have been shown by simulations (Man, Roland & Sagui 2015). Other studies have shown that the structures of the poly-Q segments, which depend on the length of the repeat track, temperature and other experiment parameters (Vitalis, Wang & Pappu 2008;Deng, Wang & Ou-Yang 2012;Buchanan et al. 2014), can exist in α-helical configuration (Kim, Chelliah, Kim, Otwinowski & Bezprozvanny 2009), loop (Kim et al. 2009), β-hairpin (Kim 2013) and βsheet (Miettinen, Knecht, Monticelli & Ignatova 2012;Buchanan et al. 2014). ...
Spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3) are two common autosomal-dominant inherited ataxia syndromes, both of which are related to the unstable expansion of tri-nucleotide CAG repeats in the coding region of the related ATXN2 and ATXN3 genes, respectively. The poly-glutamine (poly-Q) tract encoded by the CAG repeats has long been recognized as an important factor in disease pathogenesis and progress. In this study, using the I-TASSER method for 3D structure prediction, we investigated the effect of poly-Q tract enlargement on the structure and folding of ataxin-2 and ataxin-3 proteins. Our results show good agreement with the known experimental structures of the Josephin and UIM domains providing credence to the simulation results presented here, which show that the enlargement of the poly-Q region not only affects the local structure of these regions but also affects the structures of functional domains as well as the whole protein. The changes observed in the predicted models of the UIM domains in ataxin-3 when the poly-Q track is enlarged provide new insights on possible pathogenic mechanisms.
... This event dictates the structure of the initial assembly that is faithfully extended and reproduced during elongation (Fig. 6). As examined in a recent molecular dynamics study, different β-strand-based polyQ structures have distinct propensities for initiating the aggregation process (43). We propose therefore that the particular structure that we observe in the polyQ amyloid core would be uniquely capable of nucleated elongation. ...
Significance
Huntington’s disease is a devastating and incurable inherited neurodegenerative disease. Like at least eight other diseases, its primary genetic cause is the CAG repeat expansion in a specific gene. Mutant huntingtin protein undergoes misfolding and aggregation, causing degeneration of neurons through as-yet poorly understood mechanisms. Attempts to characterize the implicated protein deposits have until now had limited success. We present our structural studies of mutant huntingtin-derived protein deposits by advanced solid-state NMR spectroscopy. We determine the essential structural features of the fibrils’ rigid core, which is shown to feature intramolecular β-hairpins tied together via interdigitating extended side chains. These structural insights have direct implications for the mechanism by which the mutant protein misfolds and self-assembles.
... Three previous works have explored the aggregation properties of Q n by computer simulations. In the first of them, the focus is on the temporal stability of the structures and on the evaluation of their amyloidogenesis and fibrillation capabilites [28]. The second study explores the landscape of possible conformations by simplifying the structure of glutamine and generating a model that efficiently samples many conformations [29]. ...
Deposits of misfolded proteins in the human brain are associated with the development of many neurodegenerative diseases. Recent studies show that these proteins have common traits even at the monomer level. Among them, a polyglutamine region that is present in huntingtin is known to exhibit a correlation between the length of the chain and the severity as well as the earliness of the onset of Huntington disease. Here, we apply bias exchange molecular dynamics to generate structures of polyglutamine expansions of several lengths and characterize the resulting independent conformations. We compare the properties of these conformations to those of the standard proteins, as well as to other homopolymeric tracts. We find that, similar to the previously studied polyvaline chains, the set of possible transient folds is much broader than the set of known-to-date folds, although the conformations have different structures. We show that the mechanical stability is not related to any simple geometrical characteristics of the structures. We demonstrate that long polyglutamine expansions result in higher mechanical stability than the shorter ones. They also have a longer life span and are substantially more prone to form knotted structures. The knotted region has an average length of 35 residues, similar to the typical threshold for most polyglutamine-related diseases. Similarly, changes in shape and mechanical stability appear once the total length of the peptide exceeds this threshold of 35 glutamine residues. We suggest that knotted conformers may also harm the cellular machinery and thus lead to disease.
... Indeed, we have found that CCs can trigger aggregation and mediate the toxicity of polyQ-expanded proteins such as huntingtin (15). These findings revealed a critical role for CC structures in the molecular pathogenesis of polyQ-expansion diseases and have been further confirmed and extended to other polyQ and Q-rich proteins (17)(18)(19)(20)(21)(22). The association, and at times even contiguity, of CC-prone polyQ repeats with polyA and polyQA repeats raises the question whether also these latter repeats can be involved in the assembly of CC structures, thus suggesting the possibility that polyQ and polyA stretches might share a common CC structural propensity and, upon expansion, cause toxicity and disease through a unitary CC-based mechanism. ...
The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins due to genetic mutations induces protein aggregation and toxicity, causing at least eighteen human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ, and polyQA repeats have a phylogenetically graded association in human and non-human proteomes, and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form - alone or with polyQ and polyQA - CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2 - a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion - and found that the stability of its polyQ/polyA CC controls its aggregation, localization, and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases.
This study presents a numerical investigation of the dimerization of polyglutamine homo‐peptides of varying length. It employs the PRIME20 intermediate resolution protein model and studies it with a flat‐histogram type Monte Carlo simulation that gives access to the thermodynamic equilibrium of this model over the complete control parameter range (for the simulations this is temperature). For densities comparable to typical in vitro experimental conditions, this study finds that the aggregation and folding of the polyglutamine chains occur concurrently. However, as a function of chain length the sequence of establishment of intra‐ and intermolecular hydrogen bonding contacts changes. Chains longer than about N = 24 polyglutamine repeat units fold first and then aggregate. This agrees well with the experimental finding that, beyond N = 24 the single polyglutamine chain is the critical nucleus for the aggregation of amyloid fibrils. A finite size scaling of the ordering temperatures reveals that for this chain length (and longer chains) folding occurs at physiological (respectively larger) temperatures, whereas shorter chains are disordered at physiological conditions.
Aberrant protein folding known as protein misfolding is counted as one of the striking factors of neurodegenerative diseases. The extensive range of pathologies caused by protein misfolding, aggregation and subsequent accumulation are mainly classified into either gain of function diseases or loss of function diseases. In order to seek for novel strategies for treatment and diagnosis of neurodegenerative diseases, insights into the mechanism of misfolding and aggregation is essential. A comprehensive knowledge on the factors influencing misfolding and aggregation is required as well. An extensive experimental study on protein aggregation is somewhat challenging due the insoluble and noncrystalline nature of amyloid fibrils. Thus there has been a growing use of computational approaches including Thermodynamic Integration, Monte Carlo simulation, docking simulation, Molecular dynamics simulation in the study of protein misfolding and aggregation. The review presents a discussion on molecular dynamics simulation alone as to how it has emerged as a promising tool in the understanding of protein misfolding and aggregation in general, detailing upon three different aspects considering four misfold prone proteins in particular. It is noticeable that all four proteins considered in this review i.e prion, superoxide dismutase1, Huntingtin and Amyloid beta are linked to chronic neurodegenerative diseases with debilitating effects. Initially the review elaborates on the factors influencing the misfolding and aggregation. Next, it addresses our current understanding of the amyloid structures and the associated aggregation mechanisms, finally, summarizing the contribution of this computational tool in the search for therapeutic strategies against the respective protein-deposition diseases.
The accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and β-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed.
Lens crystallins are subject to various types of damage during their lifetime which triggers protein misfolding and aggregation, ultimately causing cataracts. There are several models for crystallin aggregation, but a comprehensive picture of the mechanism of cataract is still underway. The complex biomolecular interactions underlying crystallin aggregation have motivated major efforts to resolve the structural details and mechanism of aggregation using multiple biophysical techniques at different resolutions. Together, experimental and computational approaches identify and characterize both amyloidogenic and amorphous aggregates leading to an improved understanding of crystallin aggregation. A rigorous characterization of the aggregation-prone intermediates is crucial in cataract-mediated drug discovery. This Perspective summarizes recent biophysical studies on lens crystallin aggregation. We evaluate the outstanding challenges, future outlook, and rewards in this fertile field of research. With lessons learned from protein folding and multiple pathways of aggregation, we highlight the differences in the overall mechanisms of age-related and congenital cataracts. We expect that a correlation between the existing and developing biophysical techniques would provide a platform to study amyloid architecture in the eye lens and reduce the existing gaps in our understanding of crystallin biophysics.
Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N‐terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β‐sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all‐atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q27, Q27P11, Q34, Q35, Q36, Q40, Q50, and Q50P11. In the models without flanking polyPs, we noticed that the transformation of a random coil to β‐sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β‐sheet structure. When polyQ length increases, the 17 N‐terminal flanking residues are more likely to adopt a β‐sheet conformation from α‐helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.
There exits strong correlation between the extended poly-glutamines (polyQ) within exon-1 of Huntingtin protein (Htt) and age onset of Huntington’s disease (HD), however, the underlying molecular mechanism is still poorly understood. Here we apply extensive molecular dynamics simulations to study the folding of Htt-exon-1 across five different polyQ-lengths. We find an increase in secondary structure motifs at longer Q-lengths, including β-sheet content that seems to contribute to the formation of increasingly compact structures. More strikingly, these longer Q-lengths adopt super-compact structures as evidenced by a surprisingly small power-law scaling exponent (0.22) between the radius-of-gyration and Q-length that is substantially below expected values for compact globule structures (~0.33) and unstructured proteins (~0.50). Hydrogen bond analyses further revealed that the super-compact behavior of polyQ is mainly due to the “glue-like” behavior of glutamine’s sidechains with significantly more sidechain-sidechain H-bonds than regular proteins in the Protein Data Bank (PDB). The orientation of the glutamine sidechains also tend to be “buried” inside, explaining why polyQ domains are insoluble on their own.
We investigate the solution and fibril conformations and structural transitions of the polyglutamine (polyQ) peptide, D2Q10K2 (Q10), by synergistically using UV Resonance Raman (UVRR) spectroscopy and Molecular Dynamics (MD) simulations. We show that Q10 adopts two distinct, monomeric solution conformational states, a collapsed β-strand and a PPII-like structure that do not readily interconvert. This clearly indicates a high activation barrier in solution that prevents equilibration between these structures. Using metadynamics, we explore the conformational energy landscape of Q10 to investigate the physical origins of this high activation barrier. We develop new insights into the conformations and hydrogen bonding environments of the glutamine side chains in the PPII and β-strand-like conformations in solution. We also use the secondary structure-inducing cosolvent, acetonitrile, to investigate the conformations present in low dielectric constant solutions with decreased solvent-peptide hydrogen bonding. As the mole fraction of acetonitrile increases, Q10 converts from PPII-like structures into α-helix-like structures and β-sheet aggregates. Electron microscopy indicates that the aggregates prepared from these acetonitrile-rich solutions show morphologies similar to our previously observed polyQ fibrils. These aggregates re-dissolve upon the addition of water! These are the first examples of reversible fibril formation. Our monomeric Q10 peptides clearly sample broad regions of their available conformational energy landscape. The work here develops molecular-level insight into monomeric Q10 conformations and investigates the activation barriers between different monomer states and their evolution into fibrils.
Aggregates of proteins containing polyglutamine (polyQ) repeats are strongly associated with several neurodegenerative diseases. The length of the repeats correlates with the severity of the disease. Previous studies have shown that pure polyQ peptides aggregate by nucleated growth polymerization and that the size of the critical nucleus (n*) decreases from tetrameric to dimeric and monomeric as length increases from Q18 to Q26. Why the critical nucleus size changes with repeat-length has been unclear. Using AWSEM (the Associative memory, Water mediated, Structure and Energy Model), we construct the aggregation free energy landscapes for polyQ peptides of different repeat-lengths. These studies show that the monomer of the shorter repeat-length (Q20) prefers an extended conformation and that its aggregation indeed has a trimeric nucleus (n*∼ 3), while a longer repeat-length monomer (Q30) prefers a β-hairpin conformation which then aggregates in a downhill fashion at 0.1mM. For an intermediate length peptide (Q26), there is an equal preference for hairpin and extended forms in the monomer which leads to a mixed inhomogeneous nucleation mechanism for fibrils. The predicted changes of monomeric structure and nucleation mechanism are confirmed by studying the aggregation free energy profile for a polyglutamine repeat with site specific PG mutations that favor the hairpin form giving results in harmony with experiments on this system.
By the forced unfolding of polyglutamine and polyalanine homopeptides in competing α-helix and β-hairpin secondary structures, we disentangle equilibrium free-energetics from non-equilibrium dissipative effects. We find that α-helices are characterized by larger friction or dissipation upon unfolding, regardless of whether they are free-energetically preferred over β-hairpins or not. Our analysis suggests that this difference is related to the internal friction and mostly caused by the different number of intra-peptide hydrogen bonds in the α-helix and β-hairpin states.
Nine inherited neurodegenerative diseases are associated with the expansion of the CAG codon. Once the translated polyglutamine expansion becomes longer than about 36 residues, it triggers the formation of intraneural protein aggregates that display the signature of cross-beta amyloid fibrils. Here, we use fully atomistic molecular dynamics simulations to probe the structural stability and conformational dynamics of both previously proposed and new polyglutamine aggregate models. We test the relative stability of parallel and antiparallel beta sheets, and characterize possible steric interfaces between neighboring sheets, and the effects of different alignments of the side-chain carboxamide dipoles. Results indicate that: (i) different initial oligomer structures converge to crystals consistent with available diffraction data, after undergoing cooperative side-chain rotational transitions and quarter-stagger displacements on a microsecond timescale; (ii) structures previously deemed stable on a hundred nanosecond timescale are unstable over the microsecond timescale; (iii) conversely, structures previously deemed unstable did not account for the correct side-chain packing; once the correct symmetry is considered the structures become stable for over a microsecond, due to tightly interdigitated side chains, which lock into highly regular polar zippers with inter-side-chain and backbone-side-chain hydrogen bonds. With these insights, we built Q_40 monomeric models with different combinations of arc and/or hairpin turns, and tested them for stability. The stable monomers were further probed as a function of repeat length. Our results are consistent with the aggregation threshold. These results explain and reconcile previously reported experimental and model discrepancies about polyglutamine aggregate structures.
Proteins are complex, yet elegant, machines fine-tuned by evolution to properly fulfill a variety of tasks in the crowded cellular environment. These are, however, very challenging numerically due to their dimension, number of degrees of freedom and the wide range of relevant time scales. With aging, some proteins misfold and form harmful amyloid aggregates associated with multiple neurodegenerative diseases, and in particular Alzheimer’s, which challenge our society today. Here, I present the coarse-grained OPEP (Optimized Potential for Efficient peptide structure Prediction) force field and what we can learn from OPEP simulations to get insights into the self-assembly of amyloid peptides.
The disease risk and age of onset of Huntingtons disease (HD) and nine other repeat disorders, strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N-termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches, as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.
The ISA-Tab format and software suite have been developed to break the silo effect induced by technology-specific formats for a variety of data types and to better support experimental metadata tracking. Experimentalists seldom use a single technique to monitor biological signals. Providing a multi-purpose, pragmatic and accessible format that abstracts away common constructs for describing Investigations, Studies and Assays, ISA is increasingly popular. To attract further interest towards the format and extend support to ensure reproducible research and reusable data, we present the Risa package, which delivers a central component to support the ISA format by enabling effortless integration with R, the popular, open source data crunching environment.
The Risa package bridges the gap between the metadata collection and curation in an ISA-compliant way and the data analysis using the widely used statistical computing environment R. The package offers functionality for: i) parsing ISA-Tab datasets into R objects, ii) augmenting annotation with extra metadata not explicitly stated in the ISA syntax; iii) interfacing with domain specific R packages iv) suggesting potentially useful R packages available in Bioconductor for subsequent processing of the experimental data described in the ISA format; and finally v) saving back to ISA-Tab files augmented with analysis specific metadata from R. We demonstrate these features by presenting use cases for mass spectrometry data and DNA microarray data.
The Risa package is open source (with LGPL license) and freely available through Bioconductor. By making Risa available, we aim to facilitate the task of processing experimental data, encouraging a uniform representation of experimental information and results while delivering tools for ensuring traceability and provenance tracking.
The Risa package is available since Bioconductor 2.11 (version 1.0.0) and version 1.2.1 appeared in Bioconductor 2.12, both along with documentation and examples. The latest version of the code is at the development branch in Bioconductor and can also be accessed from GitHub https://github.com/ISA-tools/Risa, where the issue tracker allows users to report bugs or feature requests.
The huntingtin protein is characterized by a segment of consecutive glutamines (Q$_N$) that is responsible for its fibrillation. As with other amyloid proteins, misfolding of huntingtin is related to Huntington's disease through pathways that can involve interactions with phospholipid membranes. Experimental results suggest that the N-terminal 17-amino-acid sequence (htt$^{NT}$) positioned just before the Q$_N$ region is important for the binding of huntingtin to membranes. Through all-atom explicit solvent molecular dynamics simulations, we unveil the structure and dynamics of the htt$^{NT}$Q$_N$ fragment on a phospholipid membrane at the atomic level. We observe that the insertion dynamics of this peptide can be described by four main steps -- approach, reorganization, anchoring and insertion -- that are very diverse at the atomic level. On the membrane, the htt$^{NT}$ peptide forms a stable $\alpha$-helix essentially parallel to the membrane with its non-polar side-chains -- mainly Leu-4, Leu-7, Phe-11 and Leu-14 -- positioned in the hydrophobic core of the membrane. Salt-bridges involving Glu-5, Glu-12, Lys-6 and Lys-15, as well as hydrogen bonds involving Thr-3 and Ser-13 with the phospholipids also stabilize the structure and orientation of the htt$^{NT}$ peptide. These observations do not significantly change upon adding the Q$_N$ region whose role is rather to provide, through its hydrogen bonds with the phospholipids' head group, a stable scaffold facilitating the partitioning of the htt$^{NT}$ region in the membrane. Moreover, by staying accessible to the solvent, the amyloidogenic Q$_N$ region could also play a key role for the oligomerization of htt$^{NT}$Q$_N$ on phospholipid membranes. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Several neurodegenerative diseases are associated with the polyglutamine (polyQ) repeat disorder in which a segment of consecutive glutamines in the native protein is produced with too many glutamines. Huntington’s disease, for example, is related to the misfolding of the Huntingtin protein which occurs when the polyQ segment has more than approximately 36 glutamines. Experimentally, it is known that the polyQ segment alone aggregates into β-rich conformations such as amyloid fibrils. Its aggregation is modulated by the number of glutamine residues as well as by the surrounding amino acid sequences such as the 17-amino-acid N-terminal fragment of Huntingtin which increases the aggregation rate. Little structural information is available, however, regarding the first steps of aggregation and the atomistic mechanisms of oligomerization are yet to be described. Following previous coarse-grained replica-exchange molecular dynamics simulations that show the spontaneous formation of a nanotube consisting of two intertwined antiparallel strands (Laghaei, R.; Mousseau, N. J. Chem. Phys.2010, 132, 165102), we study this configuration and some extensions of it using all-atom explicit solvent MD simulations. We compare two different lengths for the polyQ segment, 40 and 30 glutamines, and we investigate the impact of the Huntingtin N-terminal residues (httNT). Our results show that the dimeric nanotubes can provide a building block for the formation of longer nanotubes (hexamers and octamers). These longer nanotubes are characterized by large β-sheet propensities and a small solvent exposure of the main-chain atoms. Moreover, the oligomerization between two nanotubes occurs through the formation of protein/protein H-bonds and can result in an elongation of the water-filled core. Our results also show that the httNT enhances the structural stability of the β-rich seeds, suggesting a new mechanism by which it can increase the aggregation rate of the amyloidogenic polyQ sequence.
A systematic analysis is performed on the effectiveness of removing degrees of freedom from hydrogen atoms and/or increasing hydrogen masses to increase the efficiency of molecular dynamics simulations of hydrogen-rich systems such as proteins in water. In proteins, high-frequency bond-angle vibrations involving hydrogen atoms limit the time step to 3 fs, which is already a factor of 1.5 beyond the commonly used time step of 2 fs. Removing these degrees of freedom from the system by constructing hydrogen atoms as dummy atoms, allows the time step to be increased to 7 fs, a factor of 3.5 compared with 2 fs. Additionally, a gain in simulation stability can be achieved by increasing the masses of hydrogen atoms with remaining degrees of freedom from 1 to 4 u. Increasing hydrogen mass without removing the high-frequency degrees of freedom allows the time step to be increased only to 4 fs, a factor of two, compared with 2 fs. The net gain in efficiency of sampling configurational space may be up to 15% lower than expected from the increase in time step due to the increase in viscosity and decrease in diffusion constant. In principle, introducing dummy atoms and increasing hydrogen mass do not influence thermodynamical properties of the system and dynamical properties are shown to be influenced only to a moderate degree. Comparing the maximum time step attainable with these methods (7 fs) to the time step of 2 fs that is routinely used in simulation, and taking into account the increase in viscosity and decrease in diffusion constant, we can say that a net gain in simulation efficiency of a factor of 3 to 3.5 can be achieved. (C) 1999 John Wiley & Sons, Inc.
The previously developed particle mesh Ewald method is reformulated in terms of efficient B‐spline interpolation of the structure factors. This reformulation allows a natural extension of the method to potentials of the form 1/r p with p≥1. Furthermore, efficient calculation of the virial tensor follows. Use of B‐splines in place of Lagrange interpolation leads to analytic gradients as well as a significant improvement in the accuracy. We demonstrate that arbitrary accuracy can be achieved, independent of system size N, at a cost that scales as N log(N). For biomolecular systems with many thousands of atoms this method permits the use of Ewald summation at a computational cost comparable to that of a simple truncation method of 10 Å or less.
Classical Monte Carlo simulations have been carried out for liquid water in the NPT ensemble at 25 °C and 1 atm using six of the simpler intermolecular potential functions for the water dimer: Bernal–Fowler (BF), SPC, ST2, TIPS2, TIP3P, and TIP4P. Comparisons are made with experimental thermodynamic and structural data including the recent neutron diffraction results of Thiessen and Narten. The computed densities and potential energies are in reasonable accord with experiment except for the original BF model, which yields an 18% overestimate of the density and poor structural results. The TIPS2 and TIP4P potentials yield oxygen–oxygen partial structure functions in good agreement with the neutron diffraction results. The accord with the experimental OH and HH partial structure functions is poorer; however, the computed results for these functions are similar for all the potential functions. Consequently, the discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations. Comparisons are also made for self‐diffusion coefficients obtained from molecular dynamics simulations. Overall, the SPC, ST2, TIPS2, and TIP4P models give reasonable structural and thermodynamic descriptions of liquid water and they should be useful in simulations of aqueous solutions. The simplicity of the SPC, TIPS2, and TIP4P functions is also attractive from a computational standpoint.
For molecular dynamics simulations of hydrated proteins a simple yet reliable model for the intermolecular potential for water is required. Such a model must be an effective pair potential valid for liquid densities that takes average many-body interactions into account. We have developed a three-point charge model (on hydrogen and oxygen positions) with a Lennard-Jones 6–12 potential on the oxygen positions only. Parameters for the model were determined from 12 molecular dynamics runs covering the two-dimensional parameter space of charge and oxygen repulsion. Both potential energy and pressure were required to coincide with experimental values. The model has very satisfactory properties, is easily incorporated into protein-water potentials, and requires only 0.25 sec computertime per dynamics step (for 216 molecules) on a CRAY-1 computer.
Preferential accumulation of mutant proteins in the nucleus has been suggested to be the molecular culprit that confers cellular toxicity in the neurodegenerative disorders caused by polyglutamine (polyQ) expansion. Here, we use dynamic imaging approaches, orthogonal cross-seeding, and composition analysis to examine the dynamics and structure of nuclear and cytoplasmic inclusions of atrophin-1, implicated in dentatorubropallidoluysian atrophy, a polyQ-based disease with complex clinical features. Our results reveal a large heterogeneity in the dynamics of the nuclear inclusions compared with the compact and immobile cytoplasmic aggregates. At least two types of inclusions of expanded atrophin-1 with different mobility of the molecular species and ability to exchange with the surrounding monomer pool coexist in the nucleus. Intriguingly, the enrichment of nuclear inclusions with slow dynamics parallels changes in the aggregate core architecture that are dominated by the polyQ stretch. We propose that the observed complexity in the dynamics of the nuclear inclusions provides a molecular explanation for the enhanced cellular toxicity of the nuclear aggregates in polyQ-based neurodegeneration.
Amyloid-β amyloidogenesis is reported to occur via a nucleated polymerization mechanism. If this is true, the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early nonfibrillar amyloid-β oligomers without observing amyloid fibrils, suggesting that a mechanistic revision may be needed. Here we introduce Cys-Cys-amyloid-β(1-40), which cannot bind to the latent fluorophore FlAsH as a monomer, but can bind FlAsH as an nonfibrillar oligomer or as a fibril, rendering the conjugates fluorescent. Through FlAsH monitoring of Cys-Cys-amyloid-β(1-40) aggregation, we found that amyloid-β(1-40) rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism. This methodology was used to show that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of amyloid-β amyloidogenesis while allowing fibril formation, rationalizing how low concentrations of plasmalogen ethanolamine in the brain are epidemiologically linked to Alzheimer's disease.
Because polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously, we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that over a short range of repeat lengths, from Q(23) to Q(26), the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex antiparallel β-sheet framework for the nucleus, and it further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in the size of aggregation nuclei may have a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases.
Huntington disease results from an expanded polyglutamine region in the N terminus of the huntingtin protein. HD pathology
is characterized by neuronal degeneration and protein inclusions containing N-terminal fragments of mutant huntingtin. Structural
information is minimal, though it is believed that mutant huntingtin polyglutamine adopts β structure upon conversion to a
toxic form. To this end, we designed mammalian cell expression constructs encoding compact β variants of Htt exon 1 N-terminal
fragment and tested their ability to aggregate and induce toxicity in cultured neuronal cells. In parallel, we performed molecular
dynamics simulations, which indicate that constructs with expanded polyglutamine β-strands are stabilized by main-chain hydrogen
bonding. Finally, we found a correlation between the reactivity to 3B5H10, an expanded polyglutamine antibody that recognizes
a compact β rich hairpin structure, and the ability to induce cell toxicity. These data are consistent with an important role
for a compact β structure in mutant huntingtin-induced cell toxicity.
Polyglutamine (polyQ) expansion in exon1 (XN1) of the huntingtin protein is linked to Huntington's disease. When the number of glutamines exceeds a threshold of approximately 36-40 repeats, XN1 can readily form amyloid aggregates similar to those associated with disease. Many experiments suggest that misfolding of monomeric XN1 plays an important role in the length-dependent aggregation. Elucidating the misfolding of a XN1 monomer can help determine the molecular mechanism of XN1 aggregation and potentially help develop strategies to inhibit XN1 aggregation. The flanking sequences surrounding the polyQ region can play a critical role in determining the structural rearrangement and aggregation mechanism of XN1. Few experiments have studied XN1 in its entirety, with all flanking regions. To obtain structural insights into the misfolding of XN1 toward amyloid aggregation, we perform molecular dynamics simulations on monomeric XN1 with full flanking regions, a variant missing the polyproline regions, which are hypothesized to prevent aggregation, and an isolated polyQ peptide (Q(n)). For each of these three constructs, we study glutamine repeat lengths of 23, 36, 40 and 47. We find that polyQ peptides have a positive correlation between their probability to form a beta-rich misfolded state and their expansion length. We also find that the flanking regions of XN1 affect its probability to form a beta-rich state compared to the isolated polyQ. Particularly, the polyproline regions form polyproline type II helices and decrease the probability of the polyQ region to form a beta-rich state. Additionally, by lengthening polyQ, the first N-terminal 17 residues are more likely to adopt a beta-sheet conformation rather than an alpha-helix conformation. Therefore, our molecular dynamics study provides a structural insight of XN1 misfolding and elucidates the possible role of the flanking sequences in XN1 aggregation.
Huntington disease (HD) is caused by an expansion of more than 35-40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis.
Aggregation of proteins containing polyglutamine (polyQ) expansions characterizes many neurodegenerative disorders, including Huntington's disease. Molecular chaperones modulate the aggregation and toxicity of the huntingtin (Htt) protein by an ill-defined mechanism. Here we determine how the chaperonin TRiC suppresses Htt aggregation. Unexpectedly, TRiC does not physically block the polyQ tract itself, but rather sequesters a short Htt sequence element, N-terminal to the polyQ tract, that promotes the amyloidogenic conformation. The residues of this element essential for rapid Htt aggregation are directly bound by TRiC. Our findings illustrate how molecular chaperones, which recognize hydrophobic determinants, can prevent aggregation of polar polyQ tracts associated with neurodegenerative diseases. The observation that short endogenous sequence elements can accelerate the switch of polyQ tracts to an amyloidogenic conformation provides a novel target for therapeutic strategies.
Amyloidogenic proteins and polypeptides can be divided into two structural classes, namely those which are flexible and are intrinsically disordered in their unaggregated state and those which form a compact globular structure with a well-defined tertiary fold in their normally soluble state. This review article is focused on amyloid formation by natively disordered polypeptides. Important examples of this class include islet amyloid polypeptide (IAPP, amylin), pro-IAPP processing intermediates, alpha-synuclein, the Abeta peptide, atrial natriuretic factor, calcitonin, pro-calcitonin, the medin polypeptide, as well as a range of de novo designed peptides. Amyloid formation is a complex process consisting of a lag phase during which no detectable fibril material is formed, followed by a rapid growth phase that leads to amyloid fibrils. A critical analysis of the literature suggests that a subset of intrinsically disordered polypeptides populate a helical intermediate during the lag phase. In this scenario, early formation of multimeric species is promoted by helix-helix association involving one region of the polypeptide chain which leads to a high effective concentration of an amyloidogenic sequence located in a different region of the chain. Helical intermediates appear to be particularly important in membrane-catalyzed amyloid formation and have been implicated in glycosaminoglycan mediated amyloid formation as well. There is suggestive evidence that targeting helix-helix interactions can be a viable strategy to inhibit amyloid formation. The characterization of transient helical intermediates is challenging, but new methods are being developed that offer the prospect of providing residue-specific information in real time.
Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTT(NT)) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTT(NT) peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTT(NT), in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTT(NT) cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTT(NT) and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntington's disease.
Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.
Several dominant, late-onset neurodegenerative diseases (e.g. Huntington's disease) are caused by expansion of polyglutamine (polyQ) repeats within specific proteins. The diverse, yet overlapping, pathology of these diseases could be due to novel deleterious functions unique to each protein or to a common pathophysiology mediated by the long polyQ chains themselves. By engineering Drosophila to express different polyQ peptides, we find that expanded polyQ chains alone are intrinsically cytotoxic and cause neuronal degeneration and early adult death. We further find that this intrinsic toxicity is dependent on cell type and polyQ length and that the inclusion of other amino acids modifies and reduces toxicity. This is the first in vivo evidence that polyQs, when removed from their disease gene context, cause neurotoxicity. These studies provide a basis for understanding the diverse clinical presentations in terms of the intrinsic cytotoxic effect of polyQ peptides being modulated by protein context. Parallel experiments in which cytotoxic polyQ expansions were engineered into Dishevelled, a Drosophila protein containing a naturally occurring polyQ tract, strongly suggest that the effect of a toxic polyQ peptide can be neutralized by protein context. This animal model provides a simple and effective means of screening for therapeutics that relieves the polyQ-induced lethality, independent of any particular disease gene. By quantifying the degree of lethality in several transgenic lines, we have identified a number of genetically modified strains that are suitable for eventual testing of compounds or genes that ameliorate the pathology of polyQ peptides.
A hallmark of neurodegenerative diseases caused by polyglutamine expansion is the abnormal accumulation of mutant proteins into ubiquitin-positive inclusions. The local build-up of these ubiquitinated proteins suggests that the proteasome machinery inadequately clears misfolded proteins, resulting in their increase to potentially toxic levels. Inclusions may disrupt normal cell homeostasis by sequestering vital cellular factors, such as chaperones, proteasomes and transcription components. Here, we used fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of polyglutamine-expanded ataxin1 and inclusion-associated proteins. These experiments demonstrated that at least two types of ataxin1 inclusions exist; those that undergo rapid and complete exchange with a nucleoplasmic pool and those that contain varying levels of slow-exchanging ataxin1. Slow-exchanging inclusions contain high ubiquitin levels, but surprisingly low proteasome levels, suggesting an impairment in the ability of proteasomes to recognize ubiquitinated substrates. Proteasomes and CBP remained highly dynamic components of inclusions, indicating that although enriched with ataxin1, they are not irreversibly trapped. These results redefine our perception of polyglutamine inclusions and demonstrate the usefulness of FRAP and live cell imaging to study factors that modulate their behaviour.
The formation of amyloid-like aggregates by expanded polyglutamine (polyGln) sequences is suspected to play a critical role in the neuropathology of Huntington's disease and other expanded CAG-repeat diseases. To probe the folding of the polyGln sequence in the aggregate, we replaced Gln-Gln pairs at different sequence intervals with Pro-Gly pairs, elements that are compatible with beta-turn formation and incompatible with beta-extended chain. We find that PGQ9 and PGQ10, peptides consisting of four Q9 or Q10 elements interspersed with PG elements, undergo spontaneous aggregation as efficiently as a Q45 sequence, whereas the corresponding PGQ7 and PGQ8 peptides aggregate much less readily. Furthermore, a PDGQ9 sequence containing d-prolines aggregates more efficiently than the peptide with l-prolines, consistent with beta-turn formation in aggregate structure. Introduction of one additional Pro residue in the center of a Q9 element within PGQ9 completely blocks the peptide's ability to aggregate. This strongly suggests that the Q9 elements are required to be in extended chain for efficient aggregation to occur. We determined the critical nucleus for aggregation nucleation of the PGQ9 peptide to be one, a result identical to that for unbroken polyGln sequences. The PGQN peptide aggregates are structurally quite similar to Q45 aggregates, as judged by heterologous seeding aggregation kinetics, recognition by an anti-polyGln aggregate antibody, and electron microscopy. The results suggest that polyGln aggregate structure consists of alternating elements of extended chain and turn. In the future it should be possible to conduct detailed and interpretable mutational studies in the PGQ9 background.
Expansion of poiygiutamine (poSyQ) tracts m proteins results m protein aggregation and is associated with cei death m at feast arte rseyrodegenerative diseases. Disease age of onset as eorreiated with the poSyQ insert Sengih above a critica vaiue of 35-40 gSutamines. The aggregation kinetics of isoiated poSyQ peptides in vitro aSso shows a simiiar edticaS-Sength dependence. WhiSe recersi experimenta work has provided corssiderabie insights irsto pofyQ aggregation, the molecular mecharilsm of aggregation is not weH understood. Here, using computer simiaiationis of isoaated poiyQ peptides, we show that a mechanism of aggregation is the conformatiorsai transition in a singie poiyQ peptide chairs from random coif to a parafSeS β-heix. This transition occurs selectively in peptides Songer than 37 gfytamines. fn the β-heHces observed in simyiations, aH residues adopt p-strand backbone dihedra angles, and the poypeptke chain coais around a centrai heiica axis with 18.5 ±2 residues per tym. We aiso fmd that mytant poiyQ peptides with prourse-gSycisie inserts show formation of antaparaHe β-hairpins in their ground state, ira agreement with experiments. The Sower stability of mutant β-heuces expiains their iower aggregation rates compared to wiid type. Our resuits provide a moiecuiar mechanism for poiyQ-medlated aggregation.
An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolutions using fast Fourier transforms. Timings and accuracies are presented for three large crystalline ionic systems. The Journal of Chemical Physics is copyrighted by The American Institute of Physics.
In this study, we have investigated the sampling of π-helical conformations, with i, i + 5 hydrogen bonding, using empirical force fields. From replica exchange molecular dynamics simulations of the helical peptide acetyl-(AAQAA)3-amide using the CHARMM22 force field and implicit solvent, we find rapid conversion from an initial α-helix to π-helical conformations. While this confirms similar studies of different peptides in explicit solvent, it does not agree with experimental data where π-helices are rarely observed. The sampling of π-helices is significantly diminished in favor of canonical α-helices when a newly extended CHARMM22/CMAP force field is used, where the backbone dihedral φ/ψ potential map in a vacuum is matched exactly to high-level quantum mechanical data for an alanine dipeptide model system. Energetic analysis shows that the difference between α- and π-helical conformations of the alanine dipeptide in a vacuum is 2.6 kcal/mol according to quantum mechanical calculations while both conformations are energetically equivalent in the unmodified CHARMM22 potential. Similar trends are also found in a number of other empirical force fields, suggesting that the observation of π-helical conformations in molecular dynamics simulations is mostly a force field artifact.
CHARMM27 is a widespread and popular force field for biomolecular simulation, and several recent algorithms such as implicit solvent models have been developed specifically for it. We have here implemented the CHARMM force field and all necessary extended functional forms in the GROMACS molecular simulation package, to make CHARMM-specific features available and to test them in combination with techniques for extended time steps, to make all major force fields available for comparison studies in GROMACS, and to test various solvent model optimizations, in particular the effect of Lennard-Jones interactions on hydrogens. The implementation has full support both for CHARMM-specific features such as multiple potentials over the same dihedral angle and the grid-based energy correction map on the , ψ protein backbone dihedrals, as well as all GROMACS features such as virtual hydrogen interaction sites that enable 5 fs time steps. The medium-to-long time effects of both the correction maps and virtual sites have been tested by performing a series of 100 ns simulations using different models for water representation, including comparisons between CHARMM and traditional TIP3P. Including the correction maps improves sampling of near native-state conformations in our systems, and to some extent it is even able to refine distorted protein conformations. Finally, we show that this accuracy is largely maintained with a new implicit solvent implementation that works with virtual interaction sites, which enables performance in excess of 250 ns/day for a 900-atom protein on a quad-core desktop computer.
By removing the fastest degrees of freedom, constraints allow for an increase of the time step in molecular simulations. In the last decade parallel simulations have become commonplace. However, up till now efficient parallel constraint algorithms have not been used with domain decomposition. In this paper the parallel linear constraint solver (P-LINCS) is presented, which allows the constraining of all bonds in macromolecules. Additionally the energy conservation properties of (P-)LINCS are assessed in view of improvements in the accuracy of uncoupled angle constraints and integration in single precision.
The mechanism by which polyglutamine expansion in Huntington's disease (HD) results in selective neuronal degeneration remains unclear. We previously reported that the immunohistochemical distribution of N-terminal huntingtin in HD does not correspond to the severity of neuropathology, such that significantly greater numbers of huntingtin aggregates are present within the cortex than in the striatum. We now show a dissociation between huntingtin aggregation and the selective pattern of striatal neuron loss observed in HD. Aggregate formation was predominantly observed in spared interneurons, with few or no aggregates found within vulnerable spiny striatal neurons. Multiple perikaryal aggregates were present in almost all cortical NADPH-diaphorase neurons and in approximately 50% of the spared NADPH-diaphorase striatal neurons from early grade HD cases. In severe grade HD patients, aggregates were more prominent as nuclear inclusions in NADPH-diaphorase neurons, with less perikaryal and neuropil aggregation. In contrast, nuclear or perikaryal huntingtin aggregates were present in less than 4% of the vulnerable calbindin striatal neurons in all HD cases. These findings support the hypothesis that polyglutamine aggregation may not be a predictor of cell loss. Rather than a harbinger of neuronal death, mutant huntingtin aggregation may be a cytoprotective mechanism against polyglutamine-induced neurotoxicity.
An analytical algorithm, called SETTLE, for resetting the positions and velocities to satisfy the holonomic constraints on the rigid water model is presented. This method is still based on the Cartesian coordinate system and can be used in place of SHAKE and RATTLE. We implemented this algorithm in the SPASMS package of molecular mechanics and dynamics. Several series of molecular dynamics simulations were carried out to examine the performance of the new algorithm in comparison with the original RATTLE method. It was found that SETTLE is of higher accuracy and is faster than RATTLE with reasonable tolerances by three to nine times on a scalar machine. Furthermore, the performance improvement ranged from factors of 26 to 98 on a vector machine since the method presented is not iterative. © 1992 by John Wiley & Sons, Inc.
Several neurodegenerative diseases have been found to be strongly associated with proteins containing a polyglutamine stretch which is greatly expanded from approximately 20 glutamines in normal individuals to more than 40 in affected individuals. A conformational change in the expanded polyglutamine stretch has been suggested to form the molecular basis for disease onset. Model peptides containing polyglutamine tend to aggregate and become insoluble. We have synthesized readily water-soluble monomeric peptides by flanking polyglutamine stretches with sequences rich in alanine and lysine. Circular dichroism measurements show that polyglutamine stretches of length 9 or 17 adopt a random coil configuration in aqueous solution. We think that in the disease-associated peptides for normal individuals the stretches of ∼20 glutamines are in a random coil conformation, whereas in affected individuals the polyglutamine stretch may be in some other conformation. Our method to design soluble monomeric peptides containing extended polyglutamine stretches may be generally useful in studying other highly aggregating peptides.
A new Lagrangian formulation is introduced. It can be used to make molecular dynamics (MD) calculations on systems under the most general, externally applied, conditions of stress. In this formulation the MD cell shape and size can change according to dynamical equations given by this Lagrangian. This new MD technique is well suited to the study of structural transformations in solids under external stress and at finite temperature. As an example of the use of this technique we show how a single crystal of Ni behaves under uniform uniaxial compressive and tensile loads. This work confirms some of the results of static (i.e., zero temperature) calculations reported in the literature. We also show that some results regarding the stress‐strain relation obtained by static calculations are invalid at finite temperature. We find that, under compressive loading, our model of Ni shows a bifurcation in its stress‐strain relation; this bifurcation provides a link in configuration space between cubic and hexagonal close packing. It is suggested that such a transformation could perhaps be observed experimentally under extreme conditions of shock.
The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.
Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.
Polyglutamine aggregation is associated with neurodegeneration in nine different disorders. The effects of polyglutamine length and peptide concentration on the kinetics of aggregation were previously analyzed using a homogeneous nucleation model that assumes the presence of a single bottleneck along the free energy profile G(n), where n denotes the number of polyglutamine molecules. The observation of stable, soluble oligomers as intermediates along aggregation pathways is refractory to the assumptions of homogeneous nucleation. Furthermore, the analysis of in vitro kinetic data using a specific variant of homogeneous nucleation leads to confounding observations such as fractional and/or negative values for estimates of the critical nucleus size. Here, we show that the homogeneous nucleation model is inherently robust and is unlikely to yield fractional values if the underlying process is strictly homogeneous with a free energy profile G(n) that displays a sharp maximum at n=n*, where n* corresponds to the critical nucleus. Conversely, a model that includes oligomers of different size and different potentials for supporting turnover into fibrils yields estimates of fractional and/or negative nucleus sizes when the kinetic data are analyzed using the assumption of a homogeneous process. This model provides a route to reconcile independent observations of heterogeneous distributions of oligomers and other non-fibrillar aggregates with results obtained from analysis of aggregation kinetics using the assumption of a homogeneous nucleation model. In the new model, the mechanisms of fibril assembly are governed by the relative stabilities of two types of oligomers viz., fibril-competent and fibril-incompetent oligomers, the size of the smallest fibril competent oligomer, and rates for conformational conversion within different oligomers.
The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.
Polyglutamine (polyQ) diseases are caused by an abnormal expansion of CAG repeats. While their detailed structure remains unclear, polyQ peptides assume beta-sheet structures when they aggregate. To investigate the conformational ensemble of short, monomeric polyQ peptides, which consist of 15 glutamine residues (Q(15)), we performed replica exchange molecular dynamics (REMD) simulations. We found that Q(15) can assume multiple configurations due to all of the residues affecting the formation of side-chain hydrogen bonds. Analysis of the free energy landscape reveals that Q(15) has a basin for random-coil structures and another for alpha-helix or beta-turn structures. To investigate properties of aggregated polyQ peptides, we performed multiple molecular dynamics (MMD) simulations for monomeric and oligomeric Q(15). MMD revealed that the formation of oligomers stabilizes the beta-turn structure by increasing the number of hydrogen bonds between the main chains.
The multiscale coarse-graining (MS-CG) method is used to construct solvent-free CG models for polyglutamine peptides having various repeat lengths. Because the resulting CG models have fewer degrees of freedom than a corresponding all-atom simulations, they make it possible to study the self-assembly of polyglutamines at high concentrations for the first time by allowing for better equilibration and statistical sampling that is well beyond the range achievable by all-atom models. Molecular dynamics (MD) simulations performed with these models show that polyglutamine monomers with repeat lengths < or = 28 fluctuate between their folded and unfolded states. Monomers with 32 or more residues are stable and form alpha-helix solid structures. The degree of monomer compactness increases with chain length in both cases. CG MD simulations of equilibrium polyglutamine aggregates show that even at high concentrations, the system statistically fluctuates between heterogeneous and homogeneous configurations, rather than simply aggregates. The degree of aggregation and fluctuation increases with concentration and chain length. All of these phenomena are consistent with the experimental observations and may be explained by a mechanism that the collective nonbonded interactions between polyglutamine molecules in water solution are only weakly attractive. Finally, this work demonstrates that computer simulation of polypeptides self-assembly and aggregation, which is presently beyond the reach of all-atom MD simulations, is attainable using solvent-free MS-CG models.
Expansion of polyglutamine (polyQ) beyond the pathogenic threshold (35-40 Gln) is associated with several neurodegenerative diseases including Huntington's disease, several forms of spinocerebellar ataxias and spinobulbar muscular atrophy. To determine the structure of polyglutamine aggregates we perform replica-exchange molecular dynamics simulations coupled with the optimized potential for effective peptide forcefield. Using a range of temperatures from 250 to 700 K, we study the aggregation kinetics of the polyglutamine monomer and dimer with chain lengths from 30 to 50 residues. All monomers show a similar structural change at the same temperature from alpha-helical structure to random coil, without indication of any significant beta-strand. For dimers, by contrast, starting from random structures, we observe spontaneous formation of antiparallel beta-sheets and triangular and circular beta-helical structures for polyglutamine with 40 residues in a 400 ns 50 temperature replica-exchange molecular dynamics simulation (total integrated time 20 micros). This approximately 32 A diameter structure reorganizes further into a tight antiparallel double-stranded approximately 22 A nanotube with 22 residues per turn close to Perutz' model for amyloid fibers as water-filled nanotubes. This diversity of structures suggests the existence of polymorphism for polyglutamine with possibly different pathways leading to the formation of toxic oligomers and to fibrils.
Aggregation of huntingtin protein with an expanded polyglutamine region is enhanced by its 17-residue N-terminal domain, which binds to itself and to the polyglutamine region. This enhancement is inhibited when the N-terminal domain binds to the chaperonin TRiC.
Huntington's disease is a genetic neurodegenerative disorder resulting from polyglutamine (polyQ) expansion (>36Q) within the first exon of Huntingtin (Htt) protein. We applied X-ray crystallography to determine the secondary structure of the first exon (EX1) of Htt17Q. The structure of Htt17Q-EX1 consists of an amino-terminal alpha helix, poly17Q region, and polyproline helix formed by the proline-rich region. The poly17Q region adopts multiple conformations in the structure, including alpha helix, random coil, and extended loop. The conformation of the poly17Q region is influenced by the conformation of neighboring protein regions, demonstrating the importance of the native protein context. We propose that the conformational flexibility of the polyQ region observed in our structure is a common characteristic of many amyloidogenic proteins. We further propose that the pathogenic polyQ expansion in the Htt protein increases the length of the random coil, which promotes aggregation and facilitates abnormal interactions with other proteins in cells.
Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-beta-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples alpha-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate beta-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.
The role of beta-sheets in the early stages of protein aggregation, specifically amyloid formation, remains unclear. Interpretations of kinetic data have led to a specific model for the role of beta-sheets in polyglutamine aggregation. According to this model, monomeric polyglutamine, which is intrinsically disordered, goes through a rare conversion into an ordered, metastable, beta-sheeted state that nucleates aggregation. It has also been proposed that the probability of forming the critical nucleus, a specific beta-sheet conformation for the monomer, increases with increasing chain length. Here, we test this model using molecular simulations. We quantified free energy profiles in terms of beta-content for monomeric polyglutamine as a function of chain length. In accord with estimates from experimental data, the free energy penalties for forming beta-rich states are in the 10-20 kcal/mol range. However, the length dependence of these free energy penalties does not mirror interpretations of kinetic data. In addition, although homodimerization of disordered molecules is spontaneous, the imposition of conformational restraints on polyglutamine molecules does not enhance the spontaneity of intermolecular associations. Our data lead to the proposal that beta-sheet formation is an attribute of peptide-rich phases such as high molecular weight aggregates rather than monomers or oligomers.
Polyglutamine (polyQ) beta-stranded aggregates constitute the hallmark of Huntington disease. The disease is fully penetrant when Q residues are more than 36-40 ("disease threshold"). Here, based on a molecular dynamics study on polyQ helical structures of different shapes and oligomeric states, we suggest that the stability of the aggregates increases with the number of monomers, while it is rather insensitive to the number of Qs in each monomer. However, the stability of the single monomer does depend on the number of side-chain intramolecular H-bonds, and therefore on the number of Qs. If such number is lower than that of the disease threshold, the beta-stranded monomers are unstable and hence may aggregate with lower probability, consistently with experimental findings. Our results provide a possible interpretation of the apparent polyQ length dependent-toxicity, and they do not support the so-called "structural threshold hypothesis", which supposes a transition from random coil to a beta-sheet structure only above the disease threshold.
We have developed an automatic algorithm STRIDE for protein secondary structure assignment from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information. Parameters of the pattern recognition procedure were optimized using designations provided by the crystallographers as a standard-of-truth. Comparison to the currently most widely used technique DSSP by Kabsch and Sander (Biopolymers 22:2577-2637, 1983) shows that STRIDE and DSSP assign secondary structural states in 58 and 31% of 226 protein chains in our data sample, respectively, in greater agreement with the specific residue-by-residue definitions provided by the discoverers of the structures while in 11% of the chains, the assignments are the same. STRIDE delineates every 11th helix and every 32nd strand more in accord with published assignments.
The mutations responsible for several human neurodegenerative disorders are expansions of translated CAG repeats beyond a normal size range. To address the role of repeat context, we have introduced a 146-unit CAG repeat into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt). Mutant mice express a form of the HPRT protein that contains a long polyglutamine repeat. These mice develop a phenotype similar to the human translated CAG repeat disorders. Repeat containing mice show a late onset neurological phenotype that progresses to premature death. Neuronal intranuclear inclusions are present in affected mice. Our results show that CAG repeats do not need to be located within one of the classic repeat disorder genes to have a neurotoxic effect.
Given a set of kinetic data, then, the preceding discussions suggest the following approach to its analysis. 1. For purposes of establishing the reaction, ignore the final stages and concentrate on the initial 10-20% of the reaction at first. A globally optimized model may be based on a faulty assumption for the initial steps. Thus, although the whole data set may look reasonably well fit, the reaction could be misrepresented, and thus the fit unhelpful if accuracy at the later stages has come at the expense of the initial phase of the reaction. 2. What is the time course of the initial reaction? (A) Is the reaction exponential? Exponential growth gives dramatic lag times (see Fig. 3), whereas nonexponential "lag times" have a visible signal from time 0 (i.e., Fig. 2). If the data set shows the abrupt appearance of signals after a period of quiescence, the chances are excellent that the time course is exponential. High sensitivity measurement of the signal at times during the lag phase should be used to confirm the exponential nature quantitatively. Exponential reactions mean a secondary pathway is operative. (a) A cascade (tn) can look similar to an exponential, but may proceed from a multistep single-path reaction. Thus the exponential needs to be ascertained with some accuracy. (b) It is possible that some or all of the lag results from a stochastic process, i.e., formation of a single nucleus being observed. This, however, is likely to be accompanied by a secondary process, as few techniques are sensitive enough to detect a single polymer at a time, and having one nucleus form many polymers is a hallmark of a secondary process. Thus, the reproducibility of the kinetics must be established to rule out stochastics. If data show wide variation, stochastic methods as described earlier may be employed. (c) Given a secondary process, one must separate the primary nucleation process from the secondary process (by stochastic means or by use of the product B2A, as described earlier). (B) If the reaction does not begin with an exponential, is it parabolic? If so, it falls in the general class of linear polymerizations. 3. What is the concentration dependence of the reaction(s)? This will separate nucleation processes from growth, and so on. 4. If the initial reaction is neither exponential nor parabolic, a reaction mechanism needs to be proposed and evaluated. Solving the resulting equations is best done by linearization, which has the best chance of giving equations whose solutions and their sensitivity to parameters are readily understood. If this proves fruitful, full numeric solutions may be useful. 5. At this point, the full reaction may be considered to completion. 6. The physical basis of the description (sizes of parameters and their dependencies) needs to be finally considered to ensure that the mathematical success of the description rests on tenable physical grounds.
Proteolysis of mutant huntingtin (htt) has been hypothesized to occur in Huntington's disease (HD) brains. Therefore, this in vivo study examined htt fragments in cortex and striatum of adult HD and control human brains by Western blots, using domain-specific anti-htt antibodies that recognize N- and C-terminal domains of htt (residues 181-810 and 2146-2541, respectively), as well as the 17 residues at the N terminus of htt. On the basis of the patterns of htt fragments observed, different "protease-susceptible domains" were identified for proteolysis of htt in cortex compared with striatum, suggesting that htt undergoes tissue-specific proteolysis. In cortex, htt proteolysis occurs within two different N-terminal domains, termed protease-susceptible domains "A" and "B." However, in striatum, a different pattern of fragments indicated that proteolysis of striatal htt occurred within a C-terminal domain termed "C," as well as within the N-terminal domain region designated "A". Importantly, striatum from HD brains showed elevated levels of 40-50 kDa N-terminal and 30-50 kDa C-terminal fragments compared with that of controls. Increased levels of these htt fragments may occur from a combination of enhanced production or retarded degradation of fragments. Results also demonstrated tissue-specific ubiquitination of certain htt N-terminal fragments in striatum compared with cortex. Moreover, expansions of the triplet-repeat domain of the IT15 gene encoding htt was confirmed for the HD tissue samples studied. Thus, regulated tissue-specific proteolysis and ubiquitination of htt occur in human HD brains. These results suggest that the role of huntingtin proteolysis should be explored in the pathogenic mechanisms of HD.
A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.
Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.
In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2-3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the in vitro aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37 degrees C undergo a random coil to beta-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, beta-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal/mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.
In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated beta-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel beta-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.
Computational studies of proteins based on empirical force fields represent a powerful tool to obtain structure-function relationships at an atomic level, and are central in current efforts to solve the protein folding problem. The results from studies applying these tools are, however, dependent on the quality of the force fields used. In particular, accurate treatment of the peptide backbone is crucial to achieve representative conformational distributions in simulation studies. To improve the treatment of the peptide backbone, quantum mechanical (QM) and molecular mechanical (MM) calculations were undertaken on the alanine, glycine, and proline dipeptides, and the results from these calculations were combined with molecular dynamics (MD) simulations of proteins in crystal and aqueous environments. QM potential energy maps of the alanine and glycine dipeptides at the LMP2/cc-pVxZ//MP2/6-31G* levels, where x = D, T, and Q, were determined, and are compared to available QM studies on these molecules. The LMP2/cc-pVQZ//MP2/6-31G* energy surfaces for all three dipeptides were then used to improve the MM treatment of the dipeptides. These improvements included additional parameter optimization via Monte Carlo simulated annealing and extension of the potential energy function to contain peptide backbone phi, psi dihedral crossterms or a phi, psi grid-based energy correction term. Simultaneously, MD simulations of up to seven proteins in their crystalline environments were used to validate the force field enhancements. Comparison with QM and crystallographic data showed that an additional optimization of the phi, psi dihedral parameters along with the grid-based energy correction were required to yield significant improvements over the CHARMM22 force field. However, systematic deviations in the treatment of phi and psi in the helical and sheet regions were evident. Accordingly, empirical adjustments were made to the grid-based energy correction for alanine and glycine to account for these systematic differences. These adjustments lead to greater deviations from QM data for the two dipeptides but also yielded improved agreement with experimental crystallographic data. These improvements enhance the quality of the CHARMM force field in treating proteins. This extension of the potential energy function is anticipated to facilitate improved treatment of biological macromolecules via MM approaches in general.