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钙调蛋白C末端叶在激活真核生物延伸因子2激酶中的关键作用。

The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase.

作者信息

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

机构信息

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.

出版信息

bioRxiv. 2025 May 16:2025.05.13.653565. doi: 10.1101/2025.05.13.653565.

Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01df/12132486/87f682b8c1e2/nihpp-2025.05.13.653565v1-f0001.jpg

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