Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage

Morgan, Michael T.; Jbara, Muhammad; Brik, Ashraf; Wolberger, Cynthia

doi:10.1016/bs.mie.2019.01.003

Cited by 16 publications

(18 citation statements)

References 42 publications

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“…Ubiquitinated H2B was generated by expressing and purifying H2B K120C and ubiquitin K79C, which were cross-linked with DCA as described in (44). A complete protocol for generating ubiquitinated nucleosomes can be found in (68).…”

Section: Methodsmentioning

confidence: 99%

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

Hoffmann

Rahman

Worden

et al. 2022

Preprint

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The human Mixed Lineage Leukemia-1 (MLL1) complex orchestrates methylation of histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variation in the docking of MLL1-WRAD on nucleosomes and left ambiguous portions of Ash2L and the position of DPY30. We used an integrated approach combining cryo-electron microscopy and mass spectrometry-crosslinking to determine structures of the MLL1-WRAD complex bound to ubiquitinated nucleosomes containing the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The subunits in all four states are positioned on the nucleosome in manner that is similar to a previous structure of MLL1-WRAD bound to ubiquitinated nucleosome, but that differs from structures with unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.SignificanceThe Mixed Lineage Leukemia-1 (MLL1) complex plays a role in activating transcription by methylating lysine 4 in histone H3, a reaction that is stimulated by the presence of ubiquitin conjugated to histone H2B. Recent structures of the core MLL1 complex, termed MLL1-WRAD, have revealed the existence of multiple docking states and have also left ambiguous portions of the structure. Here we combine mass spectrometry-cross linking with cryo-EM to model additional regions of the MLL1-WRAD complex and identify a series of states that light on complex assembly and the role that ubiquitin plays in orienting MLL1-WRAD on nucleosomes.

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Section: Methodsmentioning

confidence: 99%

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

Hoffmann

Rahman

Worden

et al. 2022

Preprint

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“…To generate stable linked Ub proteins, Long et al introduced the synthesis of nonhydrolyzable Ub mimics using 1,3-dichloroacetone (DCA) [ 66 , 67 ] as a crosslinker and applied it to generate monoubiqutylated histone proteins ( Figure 3 C) [ 68 ]. To connect both proteins, a Gly-to-Cys mutation at position 76 was inserted into the Ub’s C-terminus (UbG76C), while the ubiquitylation site at Lys-119 in histone H2A was mutated to Cys.…”

Section: Late-stage Cys Functionalizationmentioning

confidence: 99%

Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

Harel

Jbara

2022

Molecules

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Posttranslational modifications (PTMs) dramatically expand the functional diversity of the proteome. The precise addition and removal of PTMs appears to modulate protein structure and function and control key regulatory processes in living systems. Deciphering how particular PTMs affect protein activity is a current frontier in biology and medicine. The large number of PTMs which can appear in several distinct positions, states, and combinations makes preparing such complex analogs using conventional biological and chemical tools challenging. Strategies to access homogeneous and precisely modified proteins with desired PTMs at selected sites and in feasible quantities are critical to interpreting their molecular code. Here, we summarize recent advances in posttranslational chemical mutagenesis and late-stage functionalization chemistry to transfer novel PTM mimicry into recombinant proteins with emphasis on novel transformations.

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“…More recently, a 1,3-dichloroacetone-directed histone ubiquitylation method has been widely applied to the structure determination of the complexes of ubiquitylated nucleosomes with their interacting proteins, 78,79 such as Spt-Ada-Gcn5-Acetyltransferase (SAGA) DUB module, 71 Dot1L, [56][57][58][59][60]80 COMplex of Proteins ASsociated with Set1 (COMPASS), 81 MLL complex, 82 and PRC2 complex. 83 In this method, a non-cleavable mimic of the isopeptide is produced by substituting cysteines for the C-terminal glycine of Ub and H2BK120 and then crosslinking the two cysteines with DCA (1,3-dichloroacetone) (Figure 4).…”

Section: 3-dichloroacetone-directed Histone Ubiquitylationmentioning

confidence: 99%

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

Peng

2021

Journal of Peptide Science

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The reversible and dynamic post‐translational modifications (PTMs) of histones in eukaryotic chromatin are intimately connected to cell development and gene function, and abnormal regulation of PTMs can result in cancer and neurodegenerative diseases. Specific combinations of these modifications are mediated by a series of chromatin proteins that write, erase, and read the “histone codes,” but mechanistic studies of the precise biochemical and structural relationships between different sets of modifications and their effects on chromatin function constitute a unique challenge to canonical biochemical approaches. In the past decade, the development and application of chemical methods for investigating histone PTM crosstalks has received considerable attention in the field of chemical biology. In this review, taking the functional crosstalk between H2B ubiquitylation at Lys120 (H2BK120ub) and H3 methylation at Lys79 (H3K79me) as a typical example, we survey recent developments of different chemical methods, in particular, protein synthetic chemistry and protein‐based chemical probes, for studying the mechanism of the functional crosstalks of histone PTMs.

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Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage

Cited by 16 publications

References 42 publications

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

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