Assembling Ternary Dead-End Complex for Covalent Trapping of Protein Lysine Methyltransferases

Protein lysine methylation
is a distinct class of post-translational
modifications because it minimally alters the size and positive charge
of the lysine side chain. In cellular contexts, the human genome encodes
over 60 protein lysine methyltransferases (PKMTs), with the S-adenosyl-l-methionine (SAM) cofactor as the methyl
donor, to modify thousands of lysine sites on histones and nonhistone
targets in a highly orchestrated manner. The biological roles of protein
lysine methylation are increasingly implicated in epigenetic regulation
to define diverse cell fates, and their dysregulation is frequently
associated with developmental abnormalities and various aspects of
cancerous malignancy. However, it has been challenging to annotate
the multiple upstream methyltransferase(s) in parallel from known
methyllysine marks in the context of over 60 PKMT candidates with
redundant and cell-type-dependent activities. We therefore envisioned
the technology of Covalent Trapping of Protein Lysine Methyltransferases
(CTPM) by assembling the ternary dead-end complex of PKMTs with substrate-cofactor
surrogates. With SET-domain-containing PKMTs, the norleucine(Nle)-SAM
pair was shown to be a robust structural motif to form such dead-end
complexes, likely via harnessing the common feature of the transition
state of PKMT-catalyzed lysine methylation. Our CTPM peptidic probes
contain the Nle warhead in the place of substrate lysine, the photo-cross-linking
residue in proximity of Nle, and the terminal biotin anchor for target
enrichment. These CTPM probes, upon pairing with the SAM cofactor,
show high efficiency in trapping the upstream PKMTs of the cognate
histone and nonhistone substrates.

蛋白质赖氨酸甲基化（Protein lysine methylation）是一类独特的翻译后修饰（post-translational modifications），因其仅会极细微地改变赖氨酸侧链的体积与正电荷。在细胞环境中，人类基因组编码超过60种蛋白质赖氨酸甲基转移酶（protein lysine methyltransferases, PKMTs），以S-腺苷-L-甲硫氨酸（S-adenosyl-L-methionine, SAM）作为甲基供体，以高度协调的方式修饰组蛋白与非组蛋白靶标上的数千个赖氨酸位点。蛋白质赖氨酸甲基化的生物学功能愈发被证实与表观遗传调控密切相关，参与定义多种细胞命运；其失调常与发育异常以及癌症恶性进程的多个环节相关。然而，在超过60种具有冗余活性且依赖细胞类型的PKMT候选物中，同时从已知的甲基赖氨酸标记注释出对应的多个上游甲基转移酶，仍是一项极具挑战性的工作。为此，我们提出了利用底物-辅因子类似物组装PKMT三元死端复合物的蛋白质赖氨酸甲基转移酶共价捕获（Covalent Trapping of Protein Lysine Methyltransferases, CTPM）技术。针对含SET结构域的PKMT，正亮氨酸（norleucine, Nle）与SAM的配对已被证实是构建此类死端复合物的稳定结构基序，其机制可能是利用了PKMT催化赖氨酸甲基化反应的过渡态共性特征。我们设计的CTPM多肽探针以Nle作为反应弹头替代底物赖氨酸，在Nle邻近位置引入光交联残基，并在末端连接生物素锚定序列以实现靶标富集。这类CTPM探针与SAM辅因子结合后，可高效捕获对应组蛋白与非组蛋白底物的上游PKMT。