The SMC complex cohesin mediates interphase chromatin structural formation in eukaryotic cells through DNA loop extrusion. Here, we sought to investigate its mechanism using molecular dynamics simulations. To achieve this, we first constructed the amino-acid-residue-resolution structural models of the cohesin subunits, SMC1, SMC3, STAG1, and NIPBL. By simulating these subunits with double-stranded DNA molecules, we predicted DNA binding patches on each subunit and quantified the affinities of these patches to DNA using their dissociation rate constants as a proxy. Then, we constructed the structural model of the whole cohesin complex and mapped the predicted high-affinity DNA binding patches on the structure. From the spatial relations of the predicted patches, we identified that multiple patches on the SMC1, SMC3, STAG1, and NIPBL subunits form a DNA clamping patch group. The simulations of the whole complex with double-stranded DNA molecules suggest that this patch group facilitates DNA bending and helps capture a DNA segment in the cohesin ring formed by the SMC1 and SMC3 subunits. In previous studies, these have been identified as critical steps in DNA loop extrusion. Therefore, this study provides experimentally testable predictions of DNA binding sites implicated in previously proposed DNA loop extrusion mechanisms and highlights the essential roles of the accessory subunits STAG1 and NIPBL in the mechanism.

The phase separation model for transcription suggests that transcription factors (TFs), coactivators, and RNA polymerases form biomolecular condensates around active gene loci and regulate transcription. However, the structural details of condensates remain elusive. In this study, for Nanog, a master TF in mammalian embryonic stem cells known to form protein condensates in vitro, we examined protein structures in the condensates using residue-level coarse-grained molecular simulations. Human Nanog formed micelle-like clusters in the condensate. In the micelle-like cluster, the C-terminal disordered domains, including the tryptophan repeat (WR) regions, interacted with each other near the cluster center primarily via hydrophobic interaction. In contrast, hydrophilic disordered N-terminal and DNA-binding domains were exposed on the surface of the clusters. Electrostatic attractions of these surface residues were responsible for bridging multiple micelle-like structures in the condensate. The micelle-like structure and condensate were dynamic and liquid-like. Mutation of tryptophan residues in the WR region which was implicated to be important for a Nanog function resulted in dissolution of the Nanog condensate. Finally, to examine the impact of Nanog cluster to DNA, we added DNA fragments to the Nanog condensate. Nanog DNA-binding domains exposed to the surface of the micelle-like cluster could recruit more than one DNA fragments, making DNA-DNA distance shorter.

Residue-level coarse-grained (CG) models have become one of the most popular tools in biomolecular simulations in the trade-off between modeling accuracy and computational efficiency. To investigate large-scale biological phenomena in molecular dynamics (MD) simulations with CG models, unified treatments of proteins and nucleic acids, as well as efficient parallel computations, are indispensable. In the GENESIS MD software, we implement several residue-level CG models, covering structure-based and context-based potentials for both well-folded biomolecules and intrinsically disordered regions. An amino acid residue in protein is represented as a single CG particle centered at the Cα atom position, while a nucleotide in RNA or DNA is modeled with three beads. Then, a single CG particle represents around ten heavy atoms in both proteins and nucleic acids. The input data in CG MD simulations are treated as GROMACS-style input files generated from a newly developed toolbox, GENESIS-CG-tool. To optimize the performance in CG MD simulations, we utilize multiple neighbor lists, each of which is attached to a different nonbonded interaction potential in the cell-linked list method. We found that random number generations for Gaussian distributions in the Langevin thermostat are one of the bottlenecks in CG MD simulations. Therefore, we parallelize the computations with message-passing-interface (MPI) to improve the performance on PC clusters or supercomputers. We simulate Herpes simplex virus (HSV) type 2 B-capsid and chromatin models containing more than 1,000 nucleosomes in GENESIS as examples of large-scale biomolecular simulations with residue-level CG models. This framework extends accessible spatial and temporal scales by multi-scale simulations to study biologically relevant phenomena, such as genome-scale chromatin folding or phase-separated membrane-less condensations.

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. Finally, we find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences.

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. Based on these simulation results, we speculate that the conformational change of the hinge domain might be essential for the dsDNA binding regulation and play roles in condensin-mediated DNA-loop extrusion.

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhAC, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.

ATP-dependent chromatin remodelers are molecular machines that control genome organization by repositioning, ejecting, or editing nucleosomes, activities that confer them essential regulatory roles on gene expression and DNA replication. Here, we investigate the molecular mechanism of active nucleosome sliding by means of molecular dynamics simulations of the Snf2 remodeler translocase in complex with a nucleosome. During its inchworm motion driven by ATP consumption, the translocase overwrites the original nucleosome energy landscape via steric and electrostatic interactions to induce sliding of nucleosomal DNA unidirectionally. The sliding is initiated at the remodeler binding location via the generation of a pair of twist defects, which then spontaneously propagate to complete sliding throughout the entire nucleosome. We also reveal how remodeler mutations and DNA sequence control active nucleosome repositioning, explaining several past experimental observations. These results offer a detailed mechanistic picture of remodeling important for the complete understanding of these key biological processes.

Cytoplasmic dynein is a giant ATP-driven molecular motor that proceeds to the minus end of the microtubule (MT). Dynein hydrolyzes ATP in a ring-like structure, containing 6 AAA+ (ATPases associated with diverse cellular activities) modules, which is ~15 nm away from the MT binding domain (MTBD). This architecture implies that long-distance allosteric couplings exist between the AAA+ ring and the MTBD in order for dynein to move on the MT, although little is known about the mechanisms involved. Here, we have performed comprehensive molecular simulations of the dynein motor domain based on pre- and post- power-stroke structural information and in doing so we address the allosteric conformational changes that occur during the power-stroke and recovery-stroke processes. In the power-stroke process, the N-terminal linker movement was the prerequisite to the nucleotide-dependent AAA1 transition, from which a transition cascade propagated, on average, in a circular manner on the AAA+ ring until it reached the AAA6/C-terminal module. The recovery-stroke process was initiated by the transition of the AAA6/C-terminal, from which the transition cascade split into the two directions of the AAA+ ring, occurring both clockwise and anti-clockwise. In both processes, the MTBD conformational change was regulated by the AAA4 module and the AAA5/Strut module.

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood.Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA.The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence.A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions.On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation.Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements. Author summaryNucleosomes are fundamental units of chromatin folding consisting of double-stranded DNA wrapped ~1.7 times around a histone octamer.By densely populating the eukaryotic genome, nucleosomes enable efficient genome compaction inside the cellular nucleus.However, the portion of DNA occupied by a nucleosome can hardly be accessed by other DNA-binding proteins, obstructing fundamental cellular processes such as DNA replication and transcription.DNA compaction and access by other proteins can simultaneously be achieved via the dynamical repositioning of nucleosomes, which can slide along the DNA sequence.In this study, we developed and used coarse-grained molecular dynamics simulations to reveal the molecular details of nucleosome sliding.We find that the sliding mode is highly dependent on the underlying DNA sequence.Specifically, a sequence with a strong nucleosome positioning signal slides via large jumps by five and ten base pairs, preserving the optimal DNA bending profile.On the other hand, uniform sequences without the positioning signal slide via a screw-like motion of DNA, one base pair at the time.

Nucleosomes, basic units of chromatin, are known to show spontaneous DNA unwrapping dynamics that are crucial for transcriptional activation, but its structural details are yet to be elucidated. Here, employing a coarse-grained molecular model that captures residue-level structural details up to histone tails, we simulated equilibrium fluctuations and forced unwrapping of single nucleosomes at various conditions. The equilibrium simulations showed spontaneous unwrapping from outer DNA and subsequent rewrapping dynamics, which are in good agreement with experiments. We found several distinct partially unwrapped states of nucleosomes, as well as reversible transitions among these states. At a low salt concentration, histone tails tend to sit in the concave cleft between the histone octamer and DNA, tightening the nucleosome. At a higher salt concentration, the tails tend to bound to the outer side of DNA or be expanded outwards, which led to higher degree of unwrapping. Of the four types of histone tails, H3 and H2B tail dynamics are markedly correlated with partial unwrapping of DNA, and, moreover, their contributions were distinct. Acetylation in histone tails was simply mimicked by changing their charges, which enhanced the unwrapping, especially markedly for H3 and H2B tails.

Co-translational folding (CTF) facilitates correct folding in vivo, but its precise mechanism remains elusive. For the CTF of a three-domain protein SufI, it was reported that the translational attenuation is obligatory to acquire the functional state. Here, to gain structural insights on the underlying mechanisms, we performed comparative molecular simulations of SufI that mimic CTF as well as refolding schemes. A CTF scheme that relied on a codon-based prediction of translational rates exhibited folding probability markedly higher than that by the refolding scheme. When the CTF schedule is speeded up, the success rate dropped. These agree with experiments. Structural investigation clarified that misfolding of the middle domain was much more frequent in the refolding scheme than that in the codon-based CTF scheme. The middle domain is less stable and can fold via interactions with the folded N-terminal domain. Folding pathway networks showed the codon-based CTF gives narrower pathways to the native state than the refolding scheme.

Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through "walking" in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A "walking."