Long Kimberly J, Browning Luke S, Piserchio Andrea, Isiorho Eta A, Gadallah Mohamed I, Douangvilay Jomai, Wang Elizabeth Y, Kalugin Justin K, Brodbelt Jennifer S, Ghose Ranajeet, Dalby Kevin N
Division of Chemical Biology and Medicinal Chemistry, The University of Texas, Austin, TX 78712.
Interdisciplinary Life Sciences Graduate Program, The University of Texas, Austin, TX 78712.
bioRxiv. 2025 May 16:2025.05.13.653565. doi: 10.1101/2025.05.13.653565.
Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α-kinase family, modulates translational rates by phosphorylating eEF-2, a GTPase that facilitates the translocation of the nascent chain on the ribosome during the elongation phase of protein synthesis. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the Ca-effector protein calmodulin (CaM). CaM, which binds and allosterically activates eEF-2K in the presence of Ca, contains two structural "lobes," each with a pair of Ca-binding EF-hands. Using kinetic analysis, we demonstrate that the isolated C-terminal lobe of CaM (CaM) is sufficient to engage and fully activate eEF-2K in a Ca-dependent fashion. Genetically fusing CaM to the N-terminus of eEF-2K, upstream of its critical CaM-targeting motif (CTM) via a flexible 2-glycine linker, results in a chimeric species (C-LiNK) that is constitutively active independent of external CaM and Ca. A structure of the C-LiNK functional core reveals no significant deviation in the overall conformations of the interacting modules and orientations of key catalytic-site residues relative to the heterodimeric complex between full-length CaM and eEF-2K. These observations demonstrate that, in contrast to other CaM-regulated kinases, CaM alone is sufficient to activate eEF-2K fully. The proximity effect of CaM in the context of C-LiNK removes the requirement for external Ca, whose apparent role is to enhance the CaM-affinity of eEF-2K and drive kinase activation. The responsiveness of eEF-2K to regulatory stimuli in cells appears to be lost in C-LiNK, presumably due to its permanently "on" state.
真核生物延伸因子2激酶(eEF - 2K)是α激酶家族的成员,通过磷酸化eEF - 2来调节翻译速率,eEF - 2是一种GTP酶,在蛋白质合成的延伸阶段促进新生链在核糖体上的易位。eEF - 2K受多种细胞信号调节,其中许多信号使其对钙效应蛋白钙调蛋白(CaM)敏感。CaM在有钙的情况下结合并变构激活eEF - 2K,它包含两个结构“叶”,每个叶都有一对钙结合EF手。通过动力学分析,我们证明分离的CaM C末端叶(CaM)足以以钙依赖的方式结合并完全激活eEF - 2K。通过柔性双甘氨酸接头将CaM基因融合到eEF - 2K关键的CaM靶向基序(CTM)上游的N末端,产生一种嵌合体(C - LiNK),其组成性激活,独立于外部CaM和钙。C - LiNK功能核心的结构显示,与全长CaM和eEF - 2K之间的异二聚体复合物相比,相互作用模块的整体构象和关键催化位点残基的取向没有明显偏差。这些观察结果表明,与其他CaM调节的激酶不同,单独的CaM足以完全激活eEF - 2K。C - LiNK背景下CaM的邻近效应消除了对外部钙的需求,外部钙的明显作用是增强eEF - 2K对CaM的亲和力并驱动激酶激活。在细胞中,eEF - 2K对调节刺激的反应性在C - LiNK中似乎丧失了,可能是由于其永久的“开启”状态。