Biology subjects

Kono, H.

Publications and source records attributed to Kono, H..

10 recordsLinked to original sources

bioRxiv2026

Impact of intercalators on the properties of DNA analyzed by molecular dynamics simulations

Intercalation of small molecules between DNA base pairs affects DNA conformation, disrupting essential cellular processes including replication, transcription, and repair. We investigated conformational changes in 18-mer DNA upon intercalation of doxorubicin, SYBR Gold and YOYO-1 using extensive MD simulations. Two main patterns for the intercalation were identified: RISE-type intercalation occurs between adjacent base pairs and extends the DNA helix with decreased twist angles, while OPEN-type intercalation proceeds through base-pair opening without significant DNA extension. Kinetic analysis revealed that association rates for intercalation followed the order: first YO-moiety (mono-intercalation) > SYBR Gold > doxorubicin > YOYO-1 (bis-intercalation). Free energy landscape showed that forces at DNA termini reached up to 117 pN during stretching. Notably, base pairs adjacent to intercalators were protected from strand separation, accompanied by additional helical unwinding. MM-PBSA/GBSA analysis revealed that the driving force for intercalation is the stacking energy, and the binding affinity was highest for minor groove binding. Persistence length decreased with single molecule binding but recovered with two molecules due to their electrostatic repulsion. Mechanical properties of intercalated DNA showed position-dependence, demonstrating that multiple intercalation modes coexist in solution. The heterogeneous nature of intercalation explains why experimental measurements reflect ensemble averages rather than single binding configurations.

biophysics↗

bioRxiv2026

Molecular Crowding-Driven Nucleosome Interactions Revealed Through Single-Molecule Optical Tweezers

Molecular crowding causes the compaction of chromatin fibers, contributing to the formation of the nuclear architecture. However, the molecular mechanism of compaction under crowded conditions is not yet fully understood. In this study, we employed the single-molecule optical tweezer method to investigate the effect of molecular crowding on chromatin structure. Force-extension experiments on a 12-mer polynucleosome in the presence of different sizes and concentrations of polyethylene glycol (PEG) as a crowding agent showed that at low concentrations of low-molecular-weight (MW) PEG, the compaction of the polynucleosome was not significant. In this respect, nucleosomes predominantly remained separated, while DNA-histone interactions within individual nucleosomes were slightly stabilized. In contrast, high concentrations of high-MW PEG significantly promote internucleosomal interactions, leading to highly compact polynucleosome conformations. Under these conditions, approximately 30 pN of force was required to disrupt the internucleosomal interactions and release DNA; this force was 36% higher than that required for DNA unwrapping in the absence of PEG. These findings suggest that molecular crowding impacts cellular processes by mechanically regulating chromatin accessibility for regulatory proteins and the passage of motor molecules such as RNA polymerase. Significance StatementChromatin condensation is closely related to biological processes such as transcription and replication. Molecular crowding has recently attracted attention as a factor regulating chromatin condensation. In this study, we used the optical tweezer method to analyze the molecular mechanisms underlying chromatin condensation. We found that high-molecular weight and high-concentration crowders (polyethylene glycol) induced significant compaction, which involved internucleosomal interactions that markedly reduced DNA accessibility. Our results suggest that molecular crowding not only alters the condensation state, but also mechanically regulates chromatin accessibility.

biophysics↗

bioRxiv2025

HiC-SCA: A Spectral Clustering Method for Reliable A/B Compartment Assignment From Hi-C Data

A/B compartment analysis identifies regions of active and inactive chromatin organization from Hi-C data. We present HiC Spectral Compartment Assignment (HiC-SCA), a graph-based method that models chromatin as a weighted network and uses spectral clustering to partition chromosomes into A and B compartments. HiC-SCA includes four key improvements: a noise filter that removes low-quality data to prevent erroneous results, an orientation assignment method that correctly identifies A versus B compartments, a metric for selecting and assessing confidence in compartment assignments, and a resolution selection approach that identifies the highest resolution at which analysis can be performed for a dataset. Evaluation across 21 Hi-C datasets from diverse cell types using cross-dataset correlation demonstrates that HiC-SCA achieves superior consistency compared to an established method, producing more reproducible compartment assignments between datasets of the same cell type. HiC-SCA addresses key challenges in current A/B compartment analysis and provides researchers with robust tools for analyzing Hi-C datasets with varying quality.

genomics↗

bioRxiv2025

Structure and inhibition mechanism of the helicase-primase complex from human herpesvirus 1

Human herpesviruses (HHVs) are widespread pathogens causing severe diseases, especially in immunocompromised individuals, and are linked to neurodegenerative and autoimmune disorders. Current antiviral therapies primarily target the -subfamily, with limited options for {beta}- and {gamma}-subfamilies. The helicase-primase complex (HPC) is essential for viral DNA replication and represents a key antiviral target, but its structure and inhibition mechanisms were previously unclear. Here, cryo-EM structures of the HHV1 (-subfamily) HPC bound to single-stranded DNA and two clinical inhibitors, amenamevir and pritelivir, are reported. The HPC features flexible helicase and primase modules, with both inhibitors binding a shared allosteric pocket in the helicase module. Structural, biochemical, and molecular dynamics analyses indicate that these inhibitors lock the helicase in an open, inactive conformation. Combined molecular dynamics and fragment molecular orbital analyses explain the -subfamily selectivity and distinct antiviral spectra of these drugs. These findings provide a structural framework to guide the development of novel inhibitors targeting {beta}- and {gamma}-subfamily herpesviruses. SignificanceHerpesviruses are ubiquitous human pathogens responsible for a wide spectrum of diseases, including severe infections in immunocompromised individuals and associations with neurodegenerative and autoimmune disorders. Despite the clinical importance of these viruses, current antiviral therapies are largely limited to the -subfamily, with few effective options for {beta}- and {gamma}-subfamilies. The helicase-primase complex (HPC) is a critical enzyme for viral DNA replication and a validated antiviral target, yet its structural basis and inhibition mechanisms have remained elusive, hindering the rational development of next-generation therapeutics. This study provides the first high-resolution cryo-EM structures of the human herpesvirus 1 (HHV1) HPC bound to single-stranded DNA and two clinically relevant inhibitors, amenamevir and pritelivir. These structures reveal the modular organization of the HPC and identify a shared allosteric pocket where both inhibitors bind. Structural, biochemical, and molecular dynamics analyses demonstrate that these inhibitors lock the helicase in an open, catalytically inactive conformation, thereby blocking ATP binding and halting DNA unwinding. Fragment molecular orbital (FMO) calculations further elucidate the molecular determinants of inhibitor binding, explaining the -subfamily specificity and the distinct antiviral spectra of amenamevir and pritelivir. The integrated approach combining cryo-EM, molecular dynamics, and quantum chemical calculations not only clarifies the mechanism of action of current clinical inhibitors but also establishes a robust framework for the rational design of novel antivirals. By providing detailed insights into the structural and dynamic properties of the HPC and its inhibition, this work paves the way for the development of new drugs with tailored specificity and improved pharmacokinetic properties, potentially expanding therapeutic options to currently untreatable herpesvirus infections. These findings have broad implications for antiviral drug discovery and for understanding the molecular mechanisms underlying DNA replication in herpesviruses.

biochemistry↗

bioRxiv2022

Benchmark of force fields to characterize the intrinsically disordered R2-FUS-LC region

Amyloid fibrils formations are involved in many neurodegenerative diseases such as Alzheimers disease, Parkinson disease, Amyotrophic Lateral Sclerosis (ALS) and others. The proteins associated with the formation of amyloid fibrils are Intrinsically Disordered Proteins (IDP) in the unbound state. Nevertheless, this type of proteins can self-aggregate and form cross-{beta} amyloid fibrils structures at physiological condition. Due to the flexibility of these IDPs, no single experimental approach could completely characterize this system, especially in the unbound state. All-atom molecular dynamics (MD) simulations could be used to study the conformational ensemble of IDPs. Unfortunately, force fields (FF) and water models (WM) were developed to simulate one structure of folded proteins. Recently, several FF/WM were improved to properly generate conformational ensembles of IDP. However, it is unknown if the force fields (FF) can properly reproduce the behavior of IDP and also self-aggregate in cross-{beta} amyloid fibrils structures. In this paper, we will focus of the R2 region of the FUS-LC domain (R2-FUS-LC region) which is an Intrinsically Disordered Region (IDR) of 16 residues in the unbound state but forms cross-{beta} fibrils in the bound state. For the R2-FUS-LC region, we benchmarked thirteen commonly used FFs for studying IDPs. We show that CHARMM36m (updated in 2021) with mTIP3P water model performs the best to generate extended structures and cross-{beta} amyloid fibril.

biophysics↗

bioRxiv2022

Gene mapping methodology powered by induced genome rearrangements

Phenotypic variation occurs through genome rearrangements and mutations in certain responsible genes; however, systematic gene identification methodologies based on genome rearrangements have not been fully established. Here, we explored the loci responsible for the given phenotype using the TAQing system and compared it with a conventional mutagenesis-based method. Two yeast strains with different genetic backgrounds and flocculation phenotypes were fused and genomic rearrangements were induced by transient DNA breaks. Then, selection pressure was applied and multiple mutants were generated, showing different flocculation abilities. We also raised mutants with altered cohesiveness due to spontaneous mutations during long-term recursive passages of haploid strains without TAQing treatment. Comparative genomic analysis of the TAQed mutants revealed three chromosomal regions harboring pivotal flocculation genes, whereas conventional mutagenesis generated a more diverse list of candidate loci after prolonged selection. The combined use of these approaches will accelerate the identification of genes involved in complex phenotypes.

genetics↗

bioRxiv2022

Fly-casting with ligand-sliding and orientational selection to support the complex formation of a GPCR and a middle-sized flexible molecule

To elucidate computationally a binding mechanism of a middle-sized flexible molecule, bosentan, to a GPCR protein, human endothelin receptor type B (hETB), a GA-guided multidimensional virtual-system coupled molecular dynamics (GA-mD-VcMD) simulation was performed. This method is one of generalized ensemble methods and produces a free-energy landscape of the ligand-receptor binding by searching large-scale motions accompanied with stably keeping the fragile cell-membrane structure. All molecular components (bosentan, hETB, membrane, and solvent) were represented with an all-atom model, and sampling was carried out from conformations where bosentan was distant from the binding site in the hETBs binding pocket. The deepest basin in the resultant free-energy landscape was assigned to the native-like complex conformation. The obtained binding mechanism is as follows. First, bosentan fluctuating randomly in solution is captured by a tip region of the flexible N-terminal tail of hETB via nonspecific attractive interactions (fly-casting). Bosentan then occasionally slides from the tip to root of the N-terminal tail (ligand-sliding). In this sliding, bosentan passes the gate of the binding pocket from outside to inside of the pocket with accompanying a quick reduction of the molecular orientational variety of bosentan (orientational selection). Last, in the pocket, ligand-receptor attractive native contacts are formed, and eventually the native-like complex is completed. The bosentan-captured conformations by the tip- and root-regions of the N-terminal tail correspond to two basins in the free-energy landscape, and the ligand-sliding corresponds to overcoming a free-energy barrier between the basins.

biophysics↗

bioRxiv2021

Modeling SARS-CoV-2 nsp1-5'-UTR complex via the extended ensemble simulations

Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2s own RNA evades nsp1s host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results showed that the stem loop structure of the SARS-CoV-2 RNA 5-untranslated region (SL1) is recognized by both nsp1s globular region and intrinsically disordered region. The recognition presumably enables selective translation of viral RNAs. Cluster analysis of the binding mode and detailed analysis of the binding poses revealed several residues involved in the SL1 recognition mechanism. The simulation results imply that the nsp1 C-terminal helices are lifted from the 40S ribosome upon the binding of SL1 to nsp1, unblocking translation of the viral RNA.

biophysics↗

bioRxiv2020

Structural basis of the regulation of normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression by antagonizing the repressive effect of polycomb-group proteins. Aberrant upregulation of H3K36me2, either by overexpression or point mutations of NSD2/MMSET, an H3K36 dimethyltransferase, is found in various cancers, including multiple myeloma. To understand the mechanism underlying its regulation, here we report the cryo-electron microscopy structure of the catalytic fragment of NSD2 bound to the nucleosome at 2.8 [A] resolution. The nucleosomal DNA is partially unwrapped at superhelix location +5.5, facilitating the access of NSD2 to H3K36. NSD2 interacts with DNA and H2A along with H3. The autoinhibitory loop of NSD2 changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed two oncogenic mutations, E1099K and T1150A, to aberrantly activate NSD2 by increasing its catalytic turnover but not the nucleosome affinity. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing the catalytic turnover. Our analyses would guide the development of specific inhibitors of NSD2 for the treatment of various cancers.

biochemistry↗

bioRxiv2019

Structural Studies of Overlapping Dinucleosomes in Solution

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In the present study, the solution structure of the OLDN was investigated through integration of small-angle X-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis, and searched for the conformations that well reproduced the SAXS and SANS scattering curves. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.\n\nStatement of SignificanceOverlapping dinucleosomes (OLDNs) are intermediate structures formed through nucleosome collision promoted by nucleosome remodeling factor(s). To study the solution structure of OLDNs, a structural library containing a wide variety of conformations was prepared though simulations, and the structures that well reproduced the small angle X-ray and neutron scattering data were selected from the library. Simultaneous evaluation of the conformational variation in the global OLDN structures and in the histone tails is difficult using conventional MD simulations. We overcame this problem by combining multiple simulation techniques, and showed the importance of the histone tails for stabilizing the structures of OLDNs in solution.

biophysics↗