Probing the Basis of ?-Synuclein Aggregation by Comparing Simulations to Single-Molecule Experiments.
ABSTRACT: Intrinsically disordered proteins often play an important role in protein aggregation. However, it is challenging to determine the structures and interactions that drive the early stages of aggregation because they are transient and obscured in a heterogeneous mixture of disordered states. Even computational methods are limited because the lack of ordered structure makes it difficult to ensure that the relevant conformations are sampled. We address these challenges by integrating atomistic simulations with high-resolution single-molecule measurements reported previously, using the measurements to help discern which parts of the disordered ensemble of structures in the simulations are most probable while using the simulations to identify residues and interactions that are important for oligomer stability. This approach was applied to ?-synuclein, an intrinsically disordered protein that aggregates in the context of Parkinson's disease. We simulated single-molecule pulling experiments on dimers, the minimal oligomer, and compared them to force spectroscopy measurements. Force-extension curves were simulated starting from a set of 66 structures with substantial structured content selected from the ensemble of dimer structures generated at zero force via Monte Carlo simulations. The pattern of contour length changes as the structures unfolded through intermediate states was compared to the results from optical trapping measurements on the same dimer to discern likely structures occurring in the measurements. Simulated pulling curves were generally consistent with experimental data but with a larger number of transient intermediates. We identified an ensemble of ?-rich dimer structures consistent with the experimental data from which dimer interfaces could be deduced. These results suggest specific druggable targets in the structural motifs of ?-synuclein that may help prevent the earliest steps of oligomerization.
Project description:Molecular dynamics simulations using physics-based atomistic force fields have been increasingly used to characterize the heterogeneous structural ensembles of intrinsically disordered proteins (IDPs). To evaluate the accuracy of the latest atomistic explicit-solvent force fields in modeling larger IDPs with nontrivial structural features, we focus on the 61-residue N-terminal transactivation domain (TAD) of tumor suppressor p53, an important protein in cancer biology that has been extensively studied, and abundant experimental data is available for evaluation of simulated ensembles. We performed extensive replica exchange with solute tempering simulations, in excess of 1.0 ?s/replica, to generate disordered structural ensembles of p53-TAD using six latest explicit solvent protein force fields. Multiple local and long-range structural properties, including chain dimension, residual secondary structures, and transient long-range contacts, were analyzed and compared against available experimental data. The results show that IDPs such as p53-TAD remain highly challenging for atomistic simulations due to conformational complexity and difficulty in achieving adequate convergence. Structural ensembles of p53-TAD generated using various force fields differ significantly from each other. The a99SB-disp force field demonstrates the best agreement with experimental data at all levels and proves to be suitable for simulating unbound p53-TAD and how its conformational properties may be modulated by phosphorylation and other cellular signals or cancer-associated mutations. Feasibility of such detailed structural characterization is a key step toward establishing the sequence-disordered ensemble-function-disease relationship of p53 and other biologically important IDPs.
Project description:Intrinsically disordered proteins play an important role in cellular signalling, mediated by their interactions with other biomolecules. A key question concerns the nature of their binding mechanism, and whether the bound structure is induced only by proximity to the binding partner. This is difficult to answer through experiment alone because of the very heterogeneous nature of the unbound ensemble, and the probable rapid interconversion of the various unbound structures. Here we report the most extensive set of simulations on NCBD to date: we use large-scale replica exchange molecular dynamics to explore the unbound state. An important feature of the study is the use of an atomistic force field that has been parametrised against experimental data for weakly structured peptides, together with an accurate explicit water model. Neither the force field nor the starting conformations are biased towards a particular structure. The regions of NCBD that have high helical propensity in the simulations correspond closely to helices in the 'core' unbound conformation determined by NMR, although no single member of the simulated unbound ensemble closely resembles the core conformation, or either of the two known bound conformations. We have validated the results against NMR spectroscopy and SAXS measurements, obtaining reasonable agreement. The two helices which most stabilise the binding of NCBD with ACTR are formed readily; the third helix, which is less important for binding but is involved in most of the intraprotein contacts of NCBD in the bound conformation, is formed more rarely, and tends not to coexist with the other helices. These results support a mechanism by which NCBD gains the advantages of disorder, while forming binding-competent structures in the unbound state. We obtain support for this mechanism from coarse-grained simulations of NCBD with, and without, its binding partner.
Project description:Intrinsically disordered proteins play important roles throughout biology, yet our understanding of the relationship between their sequences, structural properties, and functions remains incomplete. The dynamic nature of these proteins, however, makes them difficult to characterize structurally. Many disordered proteins can attain both compact and expanded conformations, and the level of expansion may be regulated and important for function. Experimentally, the level of compaction and shape is often determined either by small-angle x-ray scattering experiments or pulsed-field-gradient NMR diffusion measurements, which provide ensemble-averaged estimates of the radius of gyration and hydrodynamic radius, respectively. Often, these experiments are interpreted using molecular simulations or are used to validate them. We here provide, to our knowledge, a new and efficient method to calculate the hydrodynamic radius of a disordered protein chain from a model of its structural ensemble. In particular, starting from basic concepts in polymer physics, we derive a relationship between the radius of gyration of a structure and its hydrodynamic ratio, which in turn can be used, for example, to compare a simulated ensemble of conformations to NMR diffusion measurements. The relationship may also be valuable when using NMR diffusion measurements to restrain molecular simulations.
Project description:Most experimentally well-characterized single domain proteins of less than 100 residues have been found to be two-state folders. That is, only two distinct populations can explain both equilibrium and kinetic measurements. Results from single molecule force spectroscopy, where a protein is unfolded by applying a mechanical pulling force to its ends, have largely confirmed this description for proteins found to be two-state in ensemble experiments. Recently, however, stable intermediates have been reported in mechanical unfolding experiments on a cold-shock protein previously found to be a prototypical two-state folder. Here, we tackle this discrepancy using free energy landscapes and Markov state models derived from coarse-grained molecular simulations. We show that protein folding intermediates can be selectively stabilized by the pulling force and that the populations of these intermediates vary in a force-dependent manner. Our model qualitatively captures the experimental results and suggests a possible origin of the apparent discrepancy.
Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging syndrome caused by the expression and accumulation of a mutant form of lamin A, ?50 lamin A. As a component of the cell's nucleoskeleton, lamin A plays an important role in the mechanical stabilization of the nuclear envelope and in other nuclear functions. It is largely unknown how the characteristic 50 amino acid deletion affects the conformation of the mostly intrinsically disordered tail domain of lamin A. Here we perform replica exchange molecular dynamics simulations of the tail domain and determine an ensemble of semi-stable structures. Based on these structures we show that the ZMPSTE 24 cleavage site on the precursor form of the lamin A tail domain orients itself in such a way as to facilitate cleavage during the maturation process. We confirm our simulated structures by comparing the thermodynamic properties of the ensemble structures to in vitro stability measurements. Using this combination of experimental and computational techniques, we compare the size, heterogeneity of size, thermodynamic stability of the Ig-fold, as well as the mechanisms of force-induced denaturation. Our data shows that the ?50 lamin A tail domain is more compact and displays less heterogeneity than the mature lamin A tail domain. Altogether these results suggest that the altered structure and stability of the tail domain can explain changed protein-protein and protein-DNA interactions and may represent an etiology of the disease. Also, this study provides the first molecular structure(s) of the lamin A tail domain, which is confirmed by thermodynamic tests in experiment.
Project description:Classical cadherin cell-cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i.e., adhesive interactions that are insensitive to force). However, the biophysical mechanism for ideal bond formation is unknown. Here, we integrate single-molecule force measurements with coarse-grained and atomistic simulations to resolve the mechanistic basis for cadherin ideal bond formation. Using simulations, we predict the energy landscape for cadherin adhesion, the transition pathways for interconversion between X-dimers and strand-swap dimers, and the cadherin structures that form ideal bonds. Based on these predictions, we engineer cadherin mutants that promote or inhibit ideal bond formation and measure their force-dependent kinetics using single-molecule force-clamp measurements with an atomic force microscope. Our data establish that cadherins adopt an intermediate conformation as they shuttle between X-dimers and strand-swap dimers; pulling on this conformation induces a torsional motion perpendicular to the pulling direction that unbinds the proteins and forms force-independent ideal bonds. Torsional motion is blocked when cadherins associate laterally in a cis orientation, suggesting that ideal bonds may play a role in mechanically regulating cadherin clustering on cell surfaces.
Project description:This article describes results on using steered molecular dynamics (SMD) simulations and experimental single molecule force spectroscopy (SMFS) to investigate the relationship between hydrogen bonding and mechanical stability of a series of homodimeric ?-sheet mimics. The dimers consisting of 4, 6, and 8 H-bonding sites were modeled in explicit chloroform solvent and the rupture force was studied using constant velocity SMD. The role of solvent structuring on the conformation of the dimers was analyzed and showed no significant contribution of chloroform molecules in the rupture event. The simulated stability of the dimers was validated by force data obtained with atomic force microscopy (AFM)-based SMFS in toluene. The computational model for the 8H dimer also offered insight into a possible mismatched dimer intermediate that may contribute to the lower than expected mechanical stability observed by single molecule AFM force studies. In addition, atomic level analysis of the rupture mechanism verified the dependence of mechanical strength on pulling trajectory due to the directional nature of chemical bonding under an applied force. The knowledge gained from this basic study will be used to guide further design of modular polymers having folded nanostructures through strategic programming of weak, non-covalent interactions into polymer backbones.
Project description:Molecular recognition is often mediated by flexible loops that have widely fluctuating structures and are sometimes disordered, but that form particular complex structures following ligand binding. In fact, many loop structures found in the PDB database are too flexible to be determined precisely. A new loop modeling method was therefore developed using force-biased multicanonical molecular dynamics with the implicit solvent model to generate an ensemble of putative loop structures with low free energy values. The method was then used to create ensembles for several flexible loops that were compared with the corresponding NMR and X-ray structures. The induced-fit structural change of dihydrofolate reductase (DHFR) was also predicted from a structural ensemble of ligand-free M20 loop conformations and successive docking simulations.
Project description:Ensemble refinement produces structural ensembles of flexible and dynamic biomolecules by integrating experimental data and molecular simulations. Here we present two efficient numerical methods to solve the computationally challenging maximum-entropy problem arising from a Bayesian formulation of ensemble refinement. Recasting the resulting constrained weight optimization problem into an unconstrained form enables the use of gradient-based algorithms. In two complementary formulations that differ in their dimensionality, we optimize either the log-weights directly or the generalized forces appearing in the explicit analytical form of the solution. We first demonstrate the robustness, accuracy, and efficiency of the two methods using synthetic data. We then use NMR J-couplings to reweight an all-atom molecular dynamics simulation ensemble of the disordered peptide Ala-5 simulated with the AMBER99SB*-ildn-q force field. After reweighting, we find a consistent increase in the population of the polyproline-II conformations and a decrease of ?-helical-like conformations. Ensemble refinement makes it possible to infer detailed structural models for biomolecules exhibiting significant dynamics, such as intrinsically disordered proteins, by combining input from experiment and simulation in a balanced manner.
Project description:Single molecule experiments that initiate folding using mechanical force are uniquely suited to reveal the nature of populated states in the folding process. Using a strategy proposed on theoretical grounds, which calls for repeated cycling of force from high to low values using force pulses, it was demonstrated in atomic force spectroscopy (AFM) experiments that an ensemble of minimum energy compact structures (MECS) are sampled during the folding of polyubiquitin. The structures in the ensemble are mechanically resistant to a lesser extent than the native state. Remarkably, forced unfolding of the populated intermediates reveals a broad distribution of extensions including steps up to 30 nm and beyond. We show using molecular simulations that favorable interdomain interactions leading to domain swapping between adjacent ubiquitin modules results in the formation of the ensemble of MECS, whose unfolding leads to an unusually broad distribution of steps. We obtained the domain-swapped structures using coarse-grained ubiquitin dimer models by exchanging native interactions between two monomeric ubiquitin molecules. Brownian dynamics force unfolding of the proposed domain-swapped structures, with mechanical stability that is approximately 100-fold lower than the native state, gives rise to a distribution of extensions from 2 to 30 nm. Our results, which are in quantitative agreement with AFM experiments, suggest that domain swapping may be a general mechanism in the assembly of multi-sub-unit proteins.