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Amyloid fibrillization is an exceedingly complex process in which incoming peptide chains bind to the fibril while concertedly folding. The coupling between folding and binding is not fully understood. We explore the molecular pathways of association of Aβ40 monomers to fibril tips by combining time-resolved in situ scanning probe microscopy with molecular modeling. The comparison between experimental and simulation results shows that a complex supported by nonnative contacts is present in the equilibrium structure of the fibril tip and impedes fibril growth in a supersaturated solution. The unraveling of this frustrated state determines the rate of fibril growth. The kinetics of growth of freshly cut fibrils, in which the bulk fibril structure persists at the tip, complemented by molecular simulations, indicate that this frustrated complex comprises three or four monomers in nonnative conformations and likely is contained on the top of a single stack of peptide chains in the fibril structure. This pathway of fibril growth strongly deviates from the common view that the conformational transformation of each captured peptide chain is templated by the previously arrived peptide. The insights into the ensemble structure of the frustrated complex may guide the search for suppressors of Aβ fibrillization. The uncovered dynamics of coupled structuring and assembly during fibril growth are more complex than during the folding of most globular proteins, as they involve the collective motions of several peptide chains that are not guided by a funneled energy landscape. 

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology. 

Assemblies of structural maintenance of chromosomes (SMC) proteins and kleisin subunits are essential to chromosome organization and segregation across all kingdoms of life. While structural data exist for parts of the SMC−kleisin complexes, complete structures of the entire complexes have yet to be determined, making mechanistic studies difficult. Using an integrative approach that combines crystallographic structural information about the globular subdomains, along with coevolutionary information and an energy landscape optimized force field (AWSEM), we predict atomic-scale structures for several tripartite SMC−kleisin complexes, including prokaryotic condensin, eukaryotic cohesin, and eukaryotic condensin. The molecular dynamics simulations of the SMC−kleisin protein complexes suggest that these complexes exist as a broad conformational ensemble that is made up of different topological isomers. The simulations suggest a critical role for the SMC coiled-coil regions, where the coils intertwine with various linking numbers. The twist and writhe of these braided coils are coupled with the motion of the SMC head domains, suggesting that the complexes may function as topological motors. Opening, closing, and translation along the DNA of the SMC−kleisin protein complexes would allow these motors to couple to the topology of DNA when DNA is entwined with the braided coils. 

The protein p53 is a crucial tumor suppressor, often called “the guardian of the genome”; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems. 

Abstract The accurate and reliable prediction of the 3D structures of proteins and their assemblies remains difficult even though the number of solved structures soars and prediction techniques improve. In this study, a free and open access web server, AWSEM-Suite, whose goal is to predict monomeric protein tertiary structures from sequence is described. The model underlying the server’s predictions is a coarse-grained protein force field which has its roots in neural network ideas that has been optimized using energy landscape theory. Employing physically motivated potentials and knowledge-based local structure biasing terms, the addition of homologous template and co-evolutionary restraints to AWSEM-Suite greatly improves the predictive power of pure AWSEM structure prediction. From the independent evaluation metrics released in the CASP13 experiment, AWSEM-Suite proves to be a reasonably accurate algorithm for free modeling, standing at the eighth position in the free modeling category of CASP13. The AWSEM-Suite server also features a front end with a user-friendly interface. The AWSEM-Suite server is a powerful tool for predicting monomeric protein tertiary structures that is most useful when a suitable structure template is not available. The AWSEM-Suite server is freely available at: https://awsem.rice.edu.