Will Nathan, Piserchio Andrea, Snyder Isaac, Ferguson Scarlet B, Giles David H, Dalby Kevin N, Ghose Ranajeet
Department of Chemistry and Biochemistry, The City College of New York , New York, New York 10031, United States.
Division of Chemical Biology and Medicinal Chemistry, University of Texas , Austin, Texas 78712, United States.
Biochemistry. 2016 Sep 27;55(38):5377-86. doi: 10.1021/acs.biochem.6b00711. Epub 2016 Sep 14.
Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, which is predicted to contain several SEL-1-like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution nuclear magnetic resonance methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (αI) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (αII-αIV). The helical core is structurally similar to members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, αII and αIII, comprise the TPR, and the last helix, αIV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing αIV and, therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding nor isolated eEF-2K627-725 inhibits eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.
真核生物延伸因子2激酶(eEF - 2K)使其唯一已知的生理底物延伸因子2(eEF - 2)磷酸化,这会降低eEF - 2与核糖体的亲和力,并导致蛋白质翻译速率整体下降。eEF - 2K的C末端区域预计包含几个SEL - 1样螺旋重复序列(SLR),它是eEF - 2磷酸化所必需的。使用溶液核磁共振方法,我们确定了eEF - 2K极端C末端的一个99个残基片段(eEF - 2K627 - 725)的结构,该片段包含一个先前被认为对eEF - 2磷酸化至关重要的区域。eEF - 2K627 - 725包含四个螺旋,其中第一个(αI)是灵活的,并且不会稳定地堆积在由最后三个螺旋(αII - αIV)形成的有序螺旋核心上。该螺旋核心在结构上类似于包含SLR的四肽重复(TPR)家族成员。倒数第二个螺旋αII和αIII构成TPR,最后一个螺旋αIV似乎具有封端功能。eEF - 2K627 - 725的结构表明,已证明可消除eEF - 2磷酸化的C末端缺失是通过使αIV不稳定从而使螺旋核心不稳定来实现的。实际上,先前已证明可消除eEF - 2磷酸化的eEF - 2K中两个保守的C末端酪氨酸(Y712A/Y713A)的突变导致eEF - 2K627 - 725解折叠。初步功能分析表明,编码对eEF - 2结合至关重要区域的肽或分离的eEF - 2K627 - 725均不会抑制全长eEF - 2K对eEF - 2的磷酸化。综上所述,我们的数据表明,单独的eEF - 2K极端C末端区域并未为eEF - 2提供主要的对接位点。