Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Commun Biol. 2024 Jun 8;7(1):707. doi: 10.1038/s42003-024-06395-z.
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.
人类蛋白赖氨酸甲基转移酶 NSD2 催化 H3K36 的二甲基化。它在发育和疾病中具有非常重要的作用，但许多机制特征及其完整的底物蛋白谱尚不清楚。我们使用肽 SPOT 阵列甲基化测定法研究了 NSD2 的底物序列特异性，并在 H3K36 上发现了 G33（-3）和 P38（+2）之间的残基的强烈读出。出乎意料的是，我们观察到 H3K36 中的天然氨基酸残基在某些位置被不同的氨基酸残基取代。将四个优选的残基结合起来，导致了一个超底物的产生，该底物在肽和蛋白质水平上被 NSD2 更快地甲基化。分子动力学模拟表明，这种活性的增加是由于酶-肽复合物的独特超活性构象所致。为了研究 NSD2 的底物谱，我们对与特异性图谱匹配的核蛋白进行了全蛋白质组搜索，发现了 NSD2 的 22 个肽底物。在蛋白质甲基化研究中，我们鉴定出 ATRX 的 K1033 和 FANCM 的 K819 是 NSD2 的甲基化位点，并在人类细胞中证明了它们的甲基化。这两种蛋白质在 DNA 修复中都具有重要作用，这加强了 NSD2 和 H3K36 甲基化与 DNA 修复之间的联系。
