Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
Elife. 2017 Oct 24;6:e29428. doi: 10.7554/eLife.29428.
The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP's activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.
内质网（ER）定位的热休克蛋白 70 伴侣蛋白 BiP 通过与未折叠的客户蛋白结合，参与一个与 ATP 结合和水解紧密偶联的过程，有助于蛋白质折叠的内稳态。BiP 的 AMP 化与未折叠 ER 蛋白的负担之间呈负相关，这表明存在一种翻译后机制，可以根据 ER 应激水平的变化来调节 BiP 的活性，但潜在的分子细节尚不清楚。我们提出了生化和晶体学研究，表明无论结合的核苷酸的身份如何，AMP 化都会使 BiP 偏向于与结合 ATP 的伴侣蛋白通常达到的构象。AMP 化不影响 BiP 与 J 蛋白共因子之间的相互作用，但似乎会变构地损害 J 蛋白刺激的 ATP 水解，导致修饰的 BiP 无法获得与其底物的高亲和力。这些发现表明了一种分子机制，通过 AMP 化作为一种开关来使 BiP 失活，限制其与底物的相互作用，同时保存 ATP。
