The T1150A cancer mutant of the protein lysine dimethyltransferase NSD2 can introduce H3K36 trimethylation

Khella, Mina S; Schnee, Philipp; Weirich, Sara; Bui, Tuan Anh; Bröhm, Alexander; Bashtrykov, Pavel; Pleiss, Jürgen; Jeltsch, Albert

doi:10.1016/j.jbc.2023.104796

Cited by 3 publications

(6 citation statements)

References 116 publications

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“…Therefore, we tested several other PKMTs to investigate if an alternative enzyme could catalyze H4K12 monomethylation. A panel of different enzymes, NSD1 (Kudithipudi et al, 2014a ), NSD2 (Khella et al, 2023 ), SMYD2 (Weirich et al, 2020 ), SET7/9 (Dhayalan et al, 2011 ), SETD6 (Kublanovsky et al, 2023 ), SET8 (Kudithipudi et al, 2012 ), SUV39H1 (Kudithipudi et al, 2017 ) and SUV39H2 (Schuhmacher et al, 2015 ) which are all well characterized in our lab, were overexpressed and purified by affinity chromatography. SDS‐PAGE analysis indicated good yield and quality for all of them (Figure S4a ).…”

Section: Resultsmentioning

confidence: 99%

“…All cloning steps were confirmed by sequencing. The details and sources of the other PKMTs are as follows: The mouse NSD1 (aka KMT3B, amino acids 1700–1987 of UniProt 088491.1) and the GST‐tagged expression constructs of the human NSD2 (aka MMSET, amino acids 992–1240 of UniProt No O96028) were taken from (Kudithipudi et al, 2014a ; Khella et al, 2023 ). The His‐tagged full‐length SET7/9 (aka KMT7, UniProt No Q8WTS6) expression construct was taken from (Dhayalan et al, 2011 ).…”

Section: Methodsmentioning

confidence: 99%

“…Histone purification, octamer formation and nucleosome reconstitution was performed as previously described (Khella et al, 2023 ; Bröhm, 2022) Histone octamers were analyzed and purified by size exclusion chromatography (Figure S5b ). The DNA assembly with the histone octamer in the reconstituted nucleosomes was validated by electromobility gel shift assays (Figure S5c ).…”

Section: Methodsmentioning

confidence: 99%

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Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues

Weirich,

Ulu,

Chandrasekaran

et al. 2024

Protein Science

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The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1‐Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q‐ and K‐methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q‐methylation preferentially occurs in a G‐Q‐X3‐R context, while K‐methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K‐methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000‐times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues

Weirich,

Ulu,

Chandrasekaran

et al. 2024

Protein Science

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“…In order to define criteria describing the likelihood of a methyl group transfer during the MD simulations and, by this, approximate the enzymatic NSD2 activity, the geometric requirements for a transition state (TS)-like conformation were applied, which were derived from the known S N 2 geometry of methyl group transfer reactions (Supplementary Fig. 7a ) 32 , 43 , 45 . Strikingly, the complex of NSD2-ssK36 established significantly more TS-like conformations than the complex of NSD2 with H3K36 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The activating T1150A mutation in NSD2 is often found in leukemia patients 31 . This mutation also changes the product specificity from H3K36me2 to H3K36me3 32 .…”

Section: Introductionmentioning

confidence: 99%

Discovery of NSD2 non-histone substrates and design of a super-substrate

Weirich,

Kusevic,

Schnee

et al. 2024

Commun Biol

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The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

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Approaching the catalytic mechanism of protein lysine methyltransferases by biochemical and simulation techniques

Schnee,

Pleiss,

Jeltsch

2024

Critical Reviews in Biochemistry and Molecular Biology

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The T1150A cancer mutant of the protein lysine dimethyltransferase NSD2 can introduce H3K36 trimethylation

Cited by 3 publications

References 116 publications

Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues

Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues

Discovery of NSD2 non-histone substrates and design of a super-substrate

Approaching the catalytic mechanism of protein lysine methyltransferases by biochemical and simulation techniques

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