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

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ko Sato ◽  
Amarjeet Kumar ◽  
Keisuke Hamada ◽  
Chikako Okada ◽  
Asako Oguni ◽  
...  
Keyword(s):  
Histone H3 ◽  
Structural Basis ◽  
Wild Type ◽  
Cryo Electron Microscopy ◽  
Nucleosomal Dna ◽  
Oncogenic Mutations ◽  
Binding Cleft ◽  
Dynamics Simulations ◽  
Nucleosome Binding ◽  
Nucleosomal Histone

AbstractDimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. 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 catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

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

2020 ◽  
Author(s):  
Ko Sato ◽  
Amarjeet Kumar ◽  
Keisuke Hamada ◽  
Chikako Okada ◽  
Asako Oguni ◽  
...  
Keyword(s):  
Histone H3 ◽  
Point Mutations ◽  
Polycomb Group ◽  
Structural Basis ◽  
Nucleosomal Dna ◽  
Oncogenic Mutations ◽  
Repressive Effect ◽  
Binding Cleft ◽  
Dynamics Simulations ◽  
Nucleosome Binding

SummaryDimethylated 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 Å 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.

scholarly journals Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids

2020 ◽  
Vol 6 (7) ◽  
pp. eaax3157 ◽  
Author(s):  
Batuujin Burendei ◽  
Ruriko Shinozaki ◽  
Masakatsu Watanabe ◽  
Tohru Terada ◽  
Kazutoshi Tani ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Structural Basis ◽  
Wild Type ◽  
Amino Terminal ◽  
Functional Assays ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Open States ◽  
Insight Into ◽  
Junction Proteins

Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo–electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

scholarly journals Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies

2021 ◽  
Vol 7 (2) ◽  
pp. eabd4413
Author(s):  
Jung-Hoon Lee ◽  
Daniel Bollschweiler ◽  
Tillman Schäfer ◽  
Robert Huber
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Active Sites ◽  
Chromatin Modification ◽  
Structural Basis ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Multiple Contacts ◽  
Lysine Residues ◽  
Nucleosome Binding

The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from acetyl-lysine residues in histone amino-terminal tails, thereby mediating transcriptional repression. Structural makeup and mechanisms by which multisubunit HDAC complexes recognize nucleosomes remain elusive. Our cryo–electron microscopy structures of the yeast class II HDAC ensembles show that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely accessible. We also observe a tetramer of protomers, where the nucleosome binding modules are inaccessible. Structural analysis of the nucleosome-bound complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC catalysis. Moreover, it reveals how an intricate network of multiple contacts between a dimer of protomers and the nucleosome creates a platform for expansion of the HDAC activities. Our study provides comprehensive insight into the structural plasticity of the HDAC complex and its functional mechanism of chromatin modification.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

scholarly journals Direct binding of TFEα opens DNA binding cleft of RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung-Hoon Jun ◽  
Jaekyung Hyun ◽  
Jeong Seok Cha ◽  
Hoyoung Kim ◽  
Michael S. Bartlett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Transcription Bubble ◽  
Cryo Electron Microscopy ◽  
Direct Binding ◽  
Binding Cleft ◽  
Promoter Dna ◽  
Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

scholarly journals Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase

2021 ◽  
Vol 4 (4) ◽  
pp. e202000919
Author(s):  
Cheng-Han Ho ◽  
Yoshimasa Takizawa ◽  
Wataru Kobayashi ◽  
Yasuhiro Arimura ◽  
Hiroshi Kimura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Essential Role ◽  
Histone H3 ◽  
Underlying Mechanism ◽  
Structural Basis ◽  
Histone H4 ◽  
Nucleosomal Histone

SET8 is solely responsible for histone H4 lysine-20 (H4K20) monomethylation, which preferentially occurs in nucleosomal H4. However, the underlying mechanism by which SET8 specifically promotes the H4K20 monomethylation in the nucleosome has not been elucidated. Here, we report the cryo-EM structures of the human SET8–nucleosome complexes with histone H3 and the centromeric H3 variant, CENP-A. Surprisingly, we found that the overall cryo-EM structures of the SET8–nucleosome complexes are substantially different from the previous crystal structure models. In the complexes with H3 and CENP-A nucleosomes, SET8 specifically binds the nucleosomal acidic patch via an arginine anchor, composed of the Arg188 and Arg192 residues. Mutational analyses revealed that the interaction between the SET8 arginine anchor and the nucleosomal acidic patch plays an essential role in the H4K20 monomethylation activity. These results provide the groundwork for understanding the mechanism by which SET8 specifically accomplishes the H4K20 monomethylation in the nucleosome.

scholarly journals Oncogenic G12D mutation alters local conformations and dynamics of K-Ras

2017 ◽  
Author(s):  
Sezen Vatansever ◽  
Burak Erman ◽  
Zeynep H. Gümüş
Keyword(s):  
Structural Changes ◽  
Structural Basis ◽  
Local Dynamic ◽  
Wild Type ◽  
Local Conformations ◽  
Dynamics Simulations ◽  
Direct Inhibitors ◽  
Detailed Picture ◽  
Inactive Protein ◽  
Prevalent Mutation

AbstractK-Ras is the most frequently mutated oncoprotein in human cancers, and G12D is its most prevalent mutation. To understand how G12D mutation impacts K-Ras function, we need to understand how it alters the regulation of its dynamics. Here, we present local changes in K-Ras structure, conformation and dynamics upon G12D mutation, from long-timescale Molecular Dynamics simulations of active (GTP-bound) and inactive (GDP-bound) forms of wild-type and mutant K-Ras, with an integrated investigation of atomistic-level changes, local conformational shifts and correlated residue motions. Our results reveal that the local changes in K-Ras are specific to bound nucleotide (GTP or GDP), and we provide a structural basis for this. Specifically, we show that G12D mutation causes a shift in the population of local conformational states of K-Ras, especially in Switch-II (SII) and α3-helix regions, in favor of a conformation that is associated with a catalytically impaired state through structural changes; it also causes SII motions to anti-correlate with other regions. This detailed picture of G12D mutation effects on the local dynamic characteristics of both active and inactive protein helps enhance our understanding of local K-Ras dynamics, and can inform studies on the development of direct inhibitors towards the treatment of K-RasG12D-driven cancers.

scholarly journals Structural basis for backtracking by the SARS-CoV-2 replication–transcription complex

2021 ◽  
Vol 118 (19) ◽  
pp. e2102516118
Author(s):  
Brandon Malone ◽  
James Chen ◽  
Qi Wang ◽  
Eliza Llewellyn ◽  
Young Joo Choi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Structural Basis ◽  
Nucleoside Triphosphate ◽  
Cross Linking ◽  
Rna Dependent Rna Polymerase ◽  
Present Evidence ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Transcription And Replication

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

scholarly journals Structural basis of the nucleosome transition during RNA polymerase II passage

Science ◽  
2018 ◽  
Vol 362 (6414) ◽  
pp. 595-598 ◽  
Author(s):  
Tomoya Kujirai ◽  
Haruhiko Ehara ◽  
Yuka Fujino ◽  
Mikako Shirouzu ◽  
Shun-ichi Sekine ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genomic Dna ◽  
Structural Basis ◽  
Histone Octamer ◽  
Contact Sites ◽  
Cryo Electron Microscopy ◽  
Nucleosomal Dna ◽  
Foreign Dna

Genomic DNA forms chromatin, in which the nucleosome is the repeating unit. The mechanism by which RNA polymerase II (RNAPII) transcribes the nucleosomal DNA remains unclear. Here we report the cryo–electron microscopy structures of RNAPII-nucleosome complexes in which RNAPII pauses at the superhelical locations SHL(−6), SHL(−5), SHL(−2), and SHL(−1) of the nucleosome. RNAPII pauses at the major histone-DNA contact sites, and the nucleosome interactions with the RNAPII subunits stabilize the pause. These structures reveal snapshots of nucleosomal transcription, in which RNAPII gradually tears DNA from the histone surface while preserving the histone octamer. The nucleosomes in the SHL(−1) complexes are bound to a “foreign” DNA segment, which might explain the histone transfer mechanism. These results provide the foundations for understanding chromatin transcription and epigenetic regulation.

scholarly journals Molecular dynamics of C99-bound γ-secretase reveal two binding modes with distinct compactness, stability, and active-site retention: implications for Aβ production

2019 ◽  
Vol 476 (7) ◽  
pp. 1173-1189 ◽  
Author(s):  
Budheswar Dehury ◽  
Ning Tang ◽  
Kasper P. Kepp
Keyword(s):  
Structural Basis ◽  
Binding Modes ◽  
Wild Type ◽  
Dynamic Simulations ◽  
Aβ Peptides ◽  
Cryo Electron Microscopy ◽  
Compact State ◽  
Β Sheet ◽  
Aβ Production

Abstract The membrane protease γ-secretase cleaves the C99 fragment of the amyloid precursor protein, thus producing the Aβ peptides central to Alzheimer's disease. Cryo-electron microscopy has provided the topology but misses the membrane and loop parts that contribute to substrate binding. We report here an essentially complete atomic model of C99 within wild-type γ-secretase that respects all the experimental constraints and additionally describes loop, helix, and C99 substrate dynamics in a realistic all-atom membrane. Our model represents the matured auto-cleaved state required for catalysis. From two independent 500-ns molecular dynamic simulations, we identify two conformation states of C99 in equilibrium, a compact and a loose state. Our simulations provide a basis for C99 processing and Aβ formation and explain the production of longer and shorter Aβ, as the compact state retains C99 for longer and thus probably trims to shorter Aβ peptides. We expect pathogenic presenilin mutations to stabilize the loose over the compact state. The simulations detail the role of the Lys53–Lys54–Lys55 anchor for C99 binding, a loss of helicity of bound C99, and positioning of Thr48 and Leu49 leading to alternative trimming pathways on opposite sides of the C99 helix in three amino acid steps. The C99 binding topology resembles that of C83-bound γ-secretase without membrane but lacks a presenilin 1-C99 β-sheet, which could be induced by C83's stronger binding. The loose state should be selectively disfavored by γ-secretase modulators to increase C99 trimming and reduce the formation of longer Aβ, a strategy that is currently much explored but has lacked a structural basis.

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