scholarly journals Structural basis of recognition and destabilization of histone H2B ubiquitinated nucleosome by DOT1L histone H3 Lys79 methyltransferase

2018 ◽  
Author(s):  
Seongmin Jang ◽  
Chanshin Kang ◽  
Han-Sol Yang ◽  
Taeyang Jung ◽  
Hans Hebert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cross Talk ◽  
Molecular Basis ◽  
Histone H3 ◽  
Structural Basis ◽  
Histone H2b ◽  
Single Molecule Fret ◽  
Catalytic Function ◽  
Histone H3 Methylation ◽  
Hydrophobic Helix

AbstractDOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B-ubiquitination. Here, we present cryo-EM structures of DOT1L complex with unmodified and H2B-ubiquitinated nucleosomes, showing that DOT1L recognizes H2B-ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L respectively. Furthermore, the structures combined with single-molecule FRET experiment show that H2B-ubiquitination enhances a non-catalytic function of DOT1L destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

scholarly journals Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase

2019 ◽  
Vol 33 (11-12) ◽  
pp. 620-625 ◽  
Author(s):  
Seongmin Jang ◽  
Chanshin Kang ◽  
Han-Sol Yang ◽  
Taeyang Jung ◽  
Hans Hebert ◽  
...  
Keyword(s):  
Histone H3 ◽  
Structural Basis ◽  
Histone H2b

scholarly journals Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET

2020 ◽  
Vol 48 (3) ◽  
pp. 1551-1571 ◽  
Author(s):  
Kathrin Lehmann ◽  
Suren Felekyan ◽  
Ralf Kühnemuth ◽  
Mykola Dimura ◽  
Katalin Tóth ◽  
...  
Keyword(s):  
Single Molecule ◽  
Histone H3 ◽  
Time Range ◽  
Histone H2a ◽  
Histone Tails ◽  
Dna Accessibility ◽  
Single Molecule Fret ◽  
Multiple Interaction ◽  
Nucleosome Disassembly ◽  
Dynamic Transitions

Abstract Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the μs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

scholarly journals Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

2019 ◽  
Author(s):  
Zhu Liu ◽  
Xu Dong ◽  
Hua-Wei Yi ◽  
Ju Yang ◽  
Zhou Gong ◽  
...  
Keyword(s):  
Single Molecule ◽  
Complex Structure ◽  
Binding Mode ◽  
Structural Basis ◽  
Ubiquitinated Proteins ◽  
Single Molecule Fret ◽  
Compact State ◽  
A Charge ◽  
Substrate Proteins ◽  
Ubiquitinated Substrate

ABSTRACTThe interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, and it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among three distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have solved the complex structure between Rpn13NTD and K48-diUb. In the structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge reversal mutation in Rpn13NTD can weaken the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, blockage of the access of the distal Ub to Rpn13NTD with a proximity attached Ub monomer can also disrupt the interaction between Rpn13 and K48-diUb. Together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.

scholarly journals Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Keijun Kakihara ◽  
Kengo Asamizu ◽  
Kei Moritsugu ◽  
Masahide Kubo ◽  
Tetsuya Kitaguchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Membrane Trafficking ◽  
Cushing’S Disease ◽  
Molecular Basis ◽  
Binding Pocket ◽  
Cushing's Disease ◽  
Binding Motif ◽  
Single Molecule Fret ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

AbstractUbiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing’s disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645–684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing’s disease due to USP8 mutations.

scholarly journals Multiple Interaction Modes of the Nucleosomal Histone H3 N-Terminal Tail Revealed by High Precision Single-Molecule FRET

2019 ◽  
Vol 116 (3) ◽  
pp. 468a-469a ◽  
Author(s):  
Kathrin Lehmann ◽  
Suren Felekyan ◽  
Ralf Kühnemuth ◽  
Mykola Dimura ◽  
Katalin Tóth ◽  
...  
Keyword(s):  
High Precision ◽  
Single Molecule ◽  
Histone H3 ◽  
Single Molecule Fret ◽  
Multiple Interaction ◽  
Nucleosomal Histone

scholarly journals Linking Enzyme Conformational Dynamics To Catalytic Function With Single-molecule FRET

2009 ◽  
Vol 96 (3) ◽  
pp. 73a-74a
Author(s):  
Yan-Wen Tan ◽  
Jeffrey A. Hanson ◽  
Jason Brokaw ◽  
Jhih-Wei Chu ◽  
Haw Yang
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret ◽  
Catalytic Function

scholarly journals Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 inTetrahymena

2009 ◽  
Vol 284 (50) ◽  
pp. 34870-34879 ◽  
Author(s):  
Zhe Wang ◽  
Bowen Cui ◽  
Martin A. Gorovsky
Keyword(s):  
Histone H3 ◽  
Histone H2b ◽  
Histone H3 Methylation

scholarly journals Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

2020 ◽  
Author(s):  
Haruka Narita ◽  
Hiroshi Ebata ◽  
Karibu Sakai ◽  
Katsuhiko Minami ◽  
Sotaro Uemura ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Dna Sequence ◽  
Single Molecule ◽  
Fluorescence Anisotropy ◽  
Molecular Basis ◽  
Catalytic Process ◽  
Single Molecule Fret ◽  
Protospacer Adjacent Motif ◽  
A Genome ◽  
Cas9 Protein

SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRETLONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

scholarly journals Structural basis for activation of Dot1L methyltransferase on the nucleosome by histone H2BK120 ubiquitylation

2018 ◽  
Author(s):  
Cathy J Anderson ◽  
Matthew R Baird ◽  
Allen Hsu ◽  
Emily H Barbour ◽  
Yuka Koyama ◽  
...  
Keyword(s):  
Histone H3 ◽  
Point Mutations ◽  
Leukemia Cells ◽  
Structural Basis ◽  
Histone H2b ◽  
Optimal Activity ◽  
A Site ◽  
H3k79 Methylation

Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of the nucleosome, is mediated by the Dot1L methyltransferase. Dot1L activity is part of a trans-histone crosstalk pathway, requiring prior histone H2B ubiquitylation of lysine 120 (H2BK120ub) for optimal activity. However, the molecular details describing both how Dot1L binds to the nucleosome and why Dot1L is activated by H2BK120 ubiquitylation are unknown. Here we present the cryo-EM structure of Dot1L bound to a nucleosome reconstituted with a site-specifically ubiquitylated H2BK120. The structure reveals that Dot1L engages the nucleosome acidic patch using an arginine anchor and occupies a conformation poised for methylation. Ubiquitin directly interacts with Dot1L and is positioned as a clamp on the nucleosome interacting region of Dot1L. Using our structure, we identify point mutations that disrupt the nucleosome-specific and ubiquitin-dependent activities of Dot1L. This study establishes a path to better understand Dot1L function in normal and leukemia cells.

scholarly journals The molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

2021 ◽  
Author(s):  
Keijun Kakihara ◽  
Kengo Asamizu ◽  
Kei Moritsugu ◽  
Masahide Kubo ◽  
Tetsuya Kitaguchi ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Single Molecule ◽  
Membrane Trafficking ◽  
Molecular Basis ◽  
Binding Pocket ◽  
Binding Motif ◽  
Deubiquitinating Enzyme ◽  
Single Molecule Fret ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins, and mutations of the 14-3-3 binding motif in USP8 are related to Cushing′s disease. However, the molecular basis of USP8 enzyme activity regulation remains unclear. Here, we identified amino acids 645–684 of USP8 as an autoinhibitory region, which our pull-down and single-molecule FRET assay results suggested interacts with the catalytic USP domain. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 was found to inhibit USP8 enzyme activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing′s disease caused by USP8 mutations.

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