23 research outputs found
Histone chaperone Nap1 dismantles an H2A/H2B dimer from a partially unwrapped nucleosome
DNA translocases, such as RNA polymerases, inevitably collide with nucleosomes on eukaryotic chromatin. Upon these collisions, histone chaperones are suggested to facilitate nucleosome disassembly and re-assembly. In this study, by performing in vitro transcription assays and molecular simulations, we found that partial unwrapping of a nucleosome by an RNA polymerase dramatically facilitates an H2A/H2B dimer dismantling from the nucleosome by Nucleosome Assembly Protein 1 (Nap1). Furthermore, the results uncovered molecular mechanisms of Nap1 functions in which the highly acidic C-terminal flexible tails of Nap1 contribute to the H2A/H2B binding by associating with the binding interface buried and not accessible to Nap1 globular domains, supporting the penetrating fuzzy binding mechanism seemingly shared across various histone chaperones. These findings have broad implications for the mechanisms by which histone chaperones process nucleosomes upon collisions with translocases in transcription, histone recycling and nucleosomal DNA repair
The lane-switch mechanism for nucleosome repositioning by DNA translocase
Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone octamers and forms nucleosomes, translocases inevitably encounter nucleosomes. A previous study has shown that a nucleosome repositions downstream when a translocase collides with the nucleosome. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identified the lane-switch mechanism for downstream repositioning with molecular dynamics simulations and validated it with restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have broad implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling
Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy
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
ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures
筋萎縮性側索硬化症(ALS)の発症機構の一端を解明 --タンパク質の高密度な凝縮構造が鍵--. 京都大学プレスリリース. 2022-09-13.T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity
p53 dynamics upon response element recognition explored by molecular simulations
p53 is a representative transcription factor that activates multiple target genes. To realize stimulus-dependent specificities, p53 has to recognize targets with structural variety, of which molecular mechanisms are largely unknown. Here, we conducted a series of long-time scale (totally more than 100-ms) coarse-grained molecular dynamics simulations, uncovering structure and dynamics of full-length p53 tetramer that recognizes its response element (RE). We obtained structures of a full-length p53 tetramer that binds to the RE, which is strikingly different from the structure of p53 at search. These structures are not only consistent with previous low-resolution or partial structural information, but also give access to previously unreachable detail, such as the preferential distribution of intrinsically disordered regions, the contacts between core domains, the DNA bending, and the connectivity of linker regions. We also explored how the RE variation affects the structure of the p53-RE complex. Further analysis of simulation trajectories revealed how p53 finds out the RE and how post-translational modifications affect the search mechanism
p53のDNA探索と認識過程のマルチスケールシミュレーションによる研究
京都大学0048新制・課程博士博士(理学)甲第18117号理博第3995号新制||理||1576(附属図書館)30975京都大学大学院理学研究科生物科学専攻(主査)教授 高田 彰二, 教授 大野 睦人, 准教授 土井 知子学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDGA
RESPAC: Method to Determine Partial Charges in Coarse-Grained Protein Model and Its Application to DNA-Binding Proteins
While coarse-grained (CG) molecular
simulations for large biomolecular
complexes have become popular, their electrostatic treatment is often
rather simplistic. Here, for <i>C</i><sub>α</sub>-based
CG models of globular proteins, we developed a method to obtain an
optimal partial charge set and applied it to 17 proteins that bind
to DNA. The method follows the restrained electrostatic potential
(RESP) fitting method widely used for determination of atomic partial
charges in all-atom (AA) molecular mechanics. The proposed method,
called the RESPAC method, finds optimal partial charges on surface <i>C</i><sub>α</sub> CG beads so that these charges best
approximate the electrostatic potential of the AA model under a restraint
term. Comparison of the AA and CG electrostatic potentials showed
that the RESPAC charges outperformed simplistic integer-valued charges.
Then, the RESPAC method was applied to lac repressor binding to a
nonspecific DNA sequence. We found that the CG simulations correlated
well with AA molecular dynamics simulations. We also performed CG
simulations of 16 other transcription factors. The differences in
binding interfaces between nonspecific and specific DNAs were, on
average, reduced by using the RESPAC charges. Yet, for several proteins,
the nonspecific DNA binding interface was quite different from that
of the specific binding interface, which is in accord with a previous
report
Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics
Protein binding to
DNA changes the DNA’s structure, and
altered DNA structure can, in turn, modulate the dynamics of protein
binding. This mutual dependency is poorly understood. Here we investigated
dynamic couplings among protein binding to DNA, protein sliding on
DNA, and DNA bending by applying a coarse-grained simulation method
to the bacterial architectural protein HU and 14 other DNA-binding
proteins. First, we verified our method by showing that the simulated
HU exhibits a weak preference for A/T-rich regions of DNA and a much
higher affinity for gapped and nicked DNA, consistent with biochemical
experiments. The high affinity was attributed to a local DNA bend,
but not the specific chemical moiety of the gap/nick. The long-time
dynamic analysis revealed that HU sliding is associated with the movement
of the local DNA bending site. Deciphering single sliding steps, we
found the coupling between HU sliding and DNA bending is akin to neither
induced-fit nor population-shift; instead they moved concomitantly.
This is reminiscent of a cation transfer on DNA and can be viewed
as a protein version of polaron-like sliding. Interestingly, on shorter
time scales, HU paused when the DNA was highly bent at the bound position
and escaped from pauses once the DNA spontaneously returned to a less
bent structure. The HU sliding is largely regulated by DNA bending
dynamics. With 14 other proteins, we explored the generality and versatility
of the dynamic coupling and found that 6 of the 15 assayed proteins
exhibit the polaron-like sliding
p53 Searches on DNA by Rotation-Uncoupled Sliding at C‑Terminal Tails and Restricted Hopping of Core Domains
The tumor suppressor p53 is a transcription factor that
searches
its cognate sites on DNA. During the search, the roles and interplay
of its two DNA binding domains, the folded core domain and the disordered
C-terminal domain (CTD), have been controversial. Here, we performed
molecular simulations of p53 at various salt concentrations finding
that, at physiological salt concentration, p53 diffuses along nonspecific
DNA via rotation-uncoupled sliding with its CTD, whereas the core
domain repeats dissociation and association. This is in perfect agreement
with a recent single molecule experiment. In the simulation of tetrameric
full-length p53, two DNA binding domains both bound to nonspecific
DNA in a characteristic form at low salt concentration, whereas at
physiological salt concentration, only CTD kept bound to DNA and the
core domain frequently hopped on DNA. Simulations of a construct that
lacks the core domain (TetCD) clarified rotation-uncoupled diffusion
on nonspecific DNA. At low salt concentration, the diffusion constant
due to sliding was dependent on the salt concentration, which differs
from the prediction of a classic theory of transcription factors.
At physiological salt concentration, it was independent of the salt
concentration, in harmony with experiments. Moreover, we found that
the sliding via the CTD follows the helical pitch of DNA (i.e., rotation-coupled
sliding) at low salt concentration while it is virtually uncoupled
to the helical pitch, a hallmark of rotation-uncoupled sliding at
physiological salt concentration