From the Departments of Biological Sciences and.
Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607.
J Biol Chem. 2018 Aug 31;293(35):13553-13565. doi: 10.1074/jbc.RA117.000262. Epub 2018 Jul 6.
Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated "MLK3 SH3-interacting peptide" (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a β-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1-4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.
混合谱系激酶 3（MLK3；也称为 MAP3K11）是一种广泛表达于正常和癌组织中的丝氨酸/苏氨酸蛋白激酶，包括脑、肺、肝、心和骨骼肌组织。其Src 同源 3（SH3）结构域参与 MLK3 的自身抑制和与其他蛋白质的相互作用，包括病毒蛋白质。MLK3 SH3 结构域含有一个六氨基酸插入序列，对应于 n-Src 插入序列，这表明 MLK3 可能结合其他多肽。在这里，通过亲和选择噬菌体展示的组合肽文库对 MLK3 的 SH3 结构域进行了筛选，得到了一个 13 肽，命名为“MLK3 SH3 相互作用肽”（MIP）。与大多数 SH3 结构域肽配体不同，MIP 含有一个脯氨酸。MIP 结合的 SH3 结构域的 1.2 Å 晶体结构显示，该肽呈 β-发夹形状，与 1.5 Å 无配体的 SH3 结构域结构比较揭示了 SH3 中的 n-Src 环发生了 MIP 诱导的构象变化。MLK3 SH3 结构域与丙型肝炎病毒非结构 5A（NS5A）蛋白的一个典型富含脯氨酸肽的 1.5 Å 结构揭示，它和 MIP 结合 SH3 结构域的两个不同位点，但生物物理分析表明这两个肽竞争与 SH3 结合。此外，MLK1 和 MLK4 的 SH3 结构域，而不是 MLK2 的 SH3 结构域，也结合 MIP，这表明 MLK1-4 家族可能通过其 SH3 结构域受到不同的调节。总之，我们已经确定了 MLK3 SH3 结构域中的两个不同的肽结合位点，为 MLK 家族激酶的配体结合机制提供了重要的见解。
