Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
Institut Laue-Langevin, Grenoble, France.
Nat Commun. 2021 Aug 18;12(1):5004. doi: 10.1038/s41467-021-25076-7.
The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.
内质网（ER）Hsp70 伴侣蛋白 BiP 受 AMP 化调节，这是一种可逆的失活翻译后修饰。BiP 的 AMP 化和去 AMP 化均由单个 ER 定位酶 FICD 催化。本文呈现了哺乳动物 AMP 化 BiP 和 FICD 之间形成的去 AMP 化 Michaelis 复合物的晶体和溶液结构。后者通过其四肽重复结构域，结合了一个专门针对 ATP 状态 Hsp70 伴侣蛋白的表面，这解释了 FICD 对 BiP 的 ATP 结合构象的高度选择性，无论是在 AMP 化还是去 AMP 化 Thr518 时。因此，揭示的真核去 AMP 化机制，合理化了保守的 Fic 结构域 Glu234 作为一个守门残基的作用，它既抑制 AMP 化，又促进二聚体 FICD 催化的水解去 AMP 化。这些发现指出，单体化诱导的 Glu234 灵活性增加是 FICD 拮抗活性的寡聚状态依赖性开关的基础，尽管其两个底物 - 未修饰和 AMP 化 BiP 的结合方式相似。
