Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications

Niederacher, Gerhard; Urwin, Debra; Dijkwel, Yasmin; Tremethick, David J.; Rosengren, K. Johan; Becker, Christian F. W.; Conibear, Anne C.

doi:10.1039/d0cb00175a

Cited by 9 publications

(7 citation statements)

References 70 publications

(119 reference statements)

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“…This may have important implications especially if phosphorylation affects HMGB1 in a different or opposite manner than what we observed for O-GlcNAc. Importantly, not all PTMs can induce dramatic alterations to protein structure and function; for instance phosphorylation studies of the HMGB1-related protein HMGN1 have recently shown mild to neutral effects on DNA binding . Thus, understanding the direct effects of phosphorylation of HMGB1 at S100 will be a critical point of interest for future studies, as well as understanding the dynamics of these two PTMs in the context of living systems.…”

Section: Discussionmentioning

confidence: 99%

O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities

et al. 2021

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Protein O-GlcNAcylation is an essential and dynamic regulator of myriad cellular processes, including DNA replication and repair. Proteomic studies have identified the multifunctional nuclear protein HMGB1 as O-GlcNAcylated, providing a potential link between this modification and DNA damage responses. Here, we verify the protein’s endogenous modification at S100 and S107 and found that the major modification site is S100, a residue that can potentially influence HMGB1-DNA interactions. Using synthetic protein chemistry, we generated site-specifically O-GlcNAc-modified HMGB1 at S100 and characterized biochemically the effect of the sugar modification on its DNA binding activity. We found that O-GlcNAc alters HMGB1 binding to linear, nucleosomal, supercoiled, cruciform, and interstrand cross-linked damaged DNA, generally resulting in enhanced oligomerization on these DNA structures. Using cell-free extracts, we also found that O-GlcNAc reduces the ability of HMGB1 to facilitate DNA repair, resulting in error-prone processing of damaged DNA. Our results expand our understanding of the molecular consequences of O-GlcNAc and how it affects protein–DNA interfaces. Importantly, our work may also support a link between upregulated O-GlcNAc levels and increased rates of mutations in certain cancer states.

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Section: Discussionmentioning

confidence: 99%

O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities

et al. 2021

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“…EPL can be used for the ligation of isotopically 15 N-and 13 C-labeled protein fragments to unlabeled protein fragments with natural abundance isotopes. Segmental labeling is useful for heteronuclear NMR analysis of large proteins because of a significant decrease in the spectral complexity, as only the labeled protein segments are observed (Chu et al, 2020;Cowburn, Shekhtman, Xu, Ottesen, & Muir, 2004;Niederacher et al, 2021).…”

Section: Commentary Background Informationmentioning

confidence: 99%

Utilizing a Baculovirus/Insect Cell Expression System and Expressed Protein Ligation (EPL) for Protein Semisynthesis

Butts

Chu

2022

Current Protocols

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Protein semisynthesis has been used for the chemoselective linking of synthetic peptides and recombinant protein fragments to generate complete native proteins in good yield. The ability to site-selectively incorporate multiple posttranslational chemical modifications (PTMs) into proteins via this approach shows great potential for enhancing understanding of the molecular basis of protein function and regulation. Protein semisynthesis, however, often requires high expression efficiency of the recombinant protein fragments (i.e., high expression yield and ability to preserve protein biological functions), which can be hard to achieve for some human enzymes when using bacterial expression systems. Here, we describe how to use a baculovirus/insect cell expression system and a protein semisynthesis strategy known as expressed protein ligation (EPL) to produce workable levels of proteins of interest containing site-specific chemical modifications. The protocol provides detailed guidance for generating protein C-terminal thioesters for use with the EPL reaction, performing the EPL reaction, and purifying the protein ligation product. We exemplify the protocols by generating protein kinase Akt1 with site-specific phosphorylations installed into its C-terminal tail, for kinetic kinase assays. We hope these methods will help increase the use of protein semisynthesis for elucidating the posttranslational regulation of human enzymes involved in cell signaling. © 2022 Wiley Periodicals LLC Basic Protocol 1: Generation of the N-terminal protein of interest (POI) fragment containing a C-terminal thioester moiety Basic Protocol 2: Expressed protein ligation (EPL) of the protein thioester with a synthetic peptide and purification of the protein ligation product Basic Protocol 3: Semisynthesis and biochemical analysis of site-specifically phosphorylated Akt1 Keywords: cell signaling enzymes r expressed protein ligation r insect cell r post-translational modifications r protein semisynthesis How to cite this article: Butts, M., & Chu, N. (2022). Utilizing a baculovirus/insect cell expression system and expressed protein ligation (EPL) for protein semisynthesis. Current Protocols, 2, e348.

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“…83 In a further example of segmental labelling by protein semisynthesis, the effects of site-specific acetylation and phosphorylation on the nucleosome-binding protein HMGN1 were recently investigated (Figure 4c). 84 Lys acetylation is catalysed by lysine acetyltransferases (KATs) and reversed by lysine deacetylases (KDACs). 89 Both enzymatic reactions can be monitored by NMR in real time, as they exhibit characteristic features on the Lys Nα and Nε in 1 H- 15 N correlation experiments.…”

Section: Rsc Chemical Biology Accepted Manuscriptmentioning

confidence: 99%

“…Open were introduced in a peptide comprising residues 1-10 of HMGN1(1-10). 84 After ligation of the N-terminal segment to the isotope-labelled, recombinant HMGN1 segment, the product was characterised by NMR spectroscopy. 1 H- 15 N HSQC NMR experiments revealed long-range perturbations in the chemical environment of the backbone residues, even in this intrinsically disordered protein (IDP).…”

Section: Rsc Chemical Biology Accepted Manuscriptmentioning

confidence: 99%

“…The segmental labelling approach facilitated resonance assignment and led to insights into PTM-modulated structural changes and functions. 84 Another frequent PTM characterised using a combination of ligation chemistry and spectroscopic investigations is ubiquitination. Ubiquitination is the covalent attachment of ubiquitin (Ub) or polyubiquitin (polyUb) chains to the Lys ε-amino group of a target protein.…”

Section: Rsc Chemical Biology Accepted Manuscriptmentioning

confidence: 99%

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Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

2021

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Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications

Abstract: Using protein semi-synthesis, segmentally isotope-labelled variants of nucleosome-binding protein HMGN1 were generated with site-specific posttranslational modifications to explore their structural and functional effects.

Cited by 9 publications

References 70 publications

O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities

O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities

Utilizing a Baculovirus/Insect Cell Expression System and Expressed Protein Ligation (EPL) for Protein Semisynthesis

Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

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