The structure and physical properties of a packaged bacteriophage particle.

A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral parts of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While the all-atom molecular dynamics method can, in principle, elucidate the conformational changes that arise from point mutations, the applications of this method to protein condensate systems is conditioned upon the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area, and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force fields that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral components of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While all-atom molecular dynamics method can, in principle, elucidate the conformational changes responsible for the aging of the condensate, the applications of this method to protein condensate systems is conditioned by the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force field that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

Percolation transition prescribes protein size-specific barrier to passive transport through the nuclear pore complex.

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex's pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of experimentally derived NPC structures with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein transport reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistics of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

Chiral Systems Made from DNA.

The very chemical structure of DNA that enables biological heredity and evolution has non-trivial implications for the self-organization of DNA molecules into larger assemblies and provides limitless opportunities for building functional nanostructures. This progress report discusses the natural organization of DNA into chiral structures and recent advances in creating synthetic chiral systems using DNA as a building material. How nucleic acid chirality naturally comes into play in a diverse array of situations is considered first, at length scales ranging from an individual nucleotide to entire chromosomes. Thereafter, chiral liquid crystal phases formed by dense DNA mixtures are discussed, including the ongoing efforts to understand their origins. The report then summarizes recent efforts directed toward building chiral structures, and other structures of complex topology, using the principle of DNA self-assembly. Discussed last are existing and proposed functional man-made nanostructures designed to either probe or harness DNA's chirality, from plasmonics and spintronics to biosensing.

Molecular dynamics simulations of DNA-DNA and DNA-protein interactions.

The all-atom molecular dynamics method can characterize the molecular-level interactions in DNA and DNA-protein systems with unprecedented resolution. Recent advances in computational technologies have allowed the method to reveal the unbiased behavior of such systems at the microseconds time scale, whereas enhanced sampling approaches have matured enough to characterize the interaction free energy with quantitative precision. Here, we describe recent progress toward increasing the realism of such simulations by refining the accuracy of the molecular dynamics force field, and we highlight recent application of the method to systems of outstanding biological interest.

Protein unfolding by SDS: the microscopic mechanisms and the properties of the SDS-protein assembly.

The effects of detergent sodium dodecyl sulfate (SDS) on protein structure and dynamics are fundamental to the most common laboratory technique used to separate proteins and determine their molecular weights: polyacrylamide gel electrophoresis. However, the mechanism by which SDS induces protein unfolding and the microstructure of protein-SDS complexes remain largely unknown. Here, we report a detailed account of SDS-induced unfolding of two proteins-I27 domain of titin and ß-amylase-obtained through all-atom molecular dynamics simulations. Both proteins were found to spontaneously unfold in the presence of SDS at boiling water temperature on the time scale of several microseconds. The protein unfolding was found to occur via two distinct mechanisms in which specific interactions of individual SDS molecules disrupt the protein's secondary structure. In the final state of the unfolding process, the proteins are found to wrap around SDS micelles in a fluid necklace-and-beads configuration, where the number and location of bound micelles changes dynamically. The global conformation of the protein was found to correlate with the number of SDS micelles bound to it, whereas the number of SDS molecules directly bound to the protein was found to define the relaxation time scale of the unfolded protein. Our microscopic characterization of SDS-protein interactions sets the stage for future refinement of SDS-enabled protein characterization methods, including protein fingerprinting and sequencing using a solid-state nanopore.

The Oligomerization Landscape of Histones.

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)2 tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.

Molecular Mechanism of Spontaneous Nucleosome Unraveling.

Meters of DNA wrap around histone proteins to form nucleosomes and fit inside the micron-diameter nucleus. For the genetic information encoded in the DNA to become available for transcription, replication, and repair, the DNA-histone assembly must be disrupted. Experiment has indicated that the outer stretches of nucleosomal DNA "breathe" by spontaneously detaching from and reattaching to the histone core. Here, we report direct observation of spontaneous DNA breathing in atomistic molecular dynamics simulations, detailing a microscopic mechanism of the DNA breathing process. According to our simulations, the outer stretches of nucleosomal DNA detach in discrete steps involving 5 or 10 base pairs, with the detachment process being orchestrated by the motion of several conserved histone residues. The inner stretches of nucleosomal DNA are found to be more stably associated with the histone core by more abundant nonspecific DNA-protein contacts, providing a microscopic interpretation of nucleosome unraveling experiments. The CG content of nucleosomal DNA is found to anticorrelate with the extent of unwrapping, supporting the possibility that AT-rich segments may signal the start of transcription by forming less stable nucleosomes.

Promiscuous Histone Mis-Assembly Is Actively Prevented by Chaperones.

Histone proteins are essential for the organization, expression, and inheritance of genetic material for eukaryotic cells. A centromere-specific H3 histone variant, centromere protein A (CENP-A), shares about 50% amino acid sequence identity with H3. CENP-A is required for packaging the centromere and for the proper separation of chromosomes during mitosis. Despite their distinct biological functions, previously reported crystal structures of the CENP-A/H4 and H3/H4 dimers reveal a high degree of similarity. In this work, we characterize the structural dynamics of CENP-A/H4 and H3/H4 dimers based on a dual-resolution approach, using both microsecond-scale explicit-solvent all-atom and coarse-grained (CG) molecular dynamics (MD) simulations. Our data show that the H4 histone is significantly more rigid compared with the H3 histone and its variant CENP-A, hence, serving as a reinforcing structural element within the histone core. We report that the CENP-A/H4 dimer is significantly more dynamic than its canonical counterpart H3/H4, and our results provide a physical explanation for this flexibility. Further, we observe that the centromere-specific chaperone Holliday Junction Recognition Protein (HJURP) stabilizes the CENP-A/H4 dimer by forming a specific electrostatic interaction network. Finally, replacing CENP-A S68 with E68 disrupts the binding interface between CENP-A and HJURP in all-atom MD simulation, and consistently, in vivo experiments demonstrate that replacing CENP-A S68 with E68 disrupts CENP-A's localization to the centromere. Based on all our results, we propose that, during the CENP-A/H4 deposition process, the chaperone HJURP protects various substructures of the dimer, serving both as a folding and binding chaperone.

Shearing of the CENP-A dimerization interface mediates plasticity in the octameric centromeric nucleosome.

The centromeric nucleosome is a key epigenetic determinant of centromere identity and function. Consequently, deciphering how CENP-A containing nucleosomes contribute structurally to centromere function is a fundamental question in chromosome biology. Here, we performed microsecond timescale all-atom molecular dynamics (MD) simulations of CENP-A and H3 nucleosomes, and report that the octameric CENP-A core particles and nucleosomes display different dynamics from their canonical H3-containing counterparts. The most significant motion observed is within key interactions at the heart of the CENP-A octameric core, wherein shearing of contacts within the CENP-A:CENP-A' dimerization interface results in a weaker four helix bundle, and an extrusion of 10-30 bp of DNA near the pseudo-dyad. Coupled to other local and global fluctuations, the CENP-A nucleosome occupies a more rugged free energy landscape than the canonical H3 nucleosome. Taken together, our data suggest that CENP-A encodes enhanced distortability to the octameric nucleosome, which may allow for enhanced flexing of the histone core in vivo.

The Acetylation Landscape of the H4 Histone Tail: Disentangling the Interplay between the Specific and Cumulative Effects.

Histone tails, the intrinsically disordered terminal regions of histone proteins, are key modulators of the structure and dynamics of chromatin and, consequently, are central to many DNA template-directed processes including replication, repair, and transcription. Acetylation of histone tails is a major post-translational modification (PTM) involved in regulating chromatin, yet it remains unclear how acetylation modifies the disordered state of histone tails and affects their function. We investigated the consequences of increasing acetylation on the isolated H4 histone tail by characterizing the conformational ensembles of unacetylated, mono-, di-, tri-, and tetra-acetylated H4 histone tails using Replica Exchange Molecular Dynamics (REMD) simulations. We found that progressive acetylation has a cumulative effect on the H4 tail, decreasing conformational heterogeneity, increasing helical propensity, and increasing hydrogen bond occupancies. The monoacetylation of lysine 16, however, has unique and specific effects: drastically decreasing the conformational heterogeneity of the H4 tail and leading to highly localized helical secondary structure and elongated conformations. We describe how the cumulative effects of acetylation arise from the charge reduction and increased hydrophobicity associated with adding acetyl groups, while the specific effects are a consequence of steric interactions that are sequence specific. Additionally, we found that increasing the level of acetylation results in the formation of spatially clustered lysines that could serve as recognition patches for binding of chromatin regulating proteins. Hence, we explore the mechanisms by which different acetylation patterns may result in specific recognition of the H4 histone tails by protein or DNA binding partners.