Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiP’s Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.

Fic（由 cAMP 诱导的丝状化）蛋白通过翻译后修饰其蛋白质靶标来调节多种细胞信号传导事件，主要是通过添加 AMP（一磷酸腺苷）。这种修饰称为 Fic 介导的腺苷酸化或 AMPylation。我们之前报道过人类 Fic 蛋白 HYPE/FicD 是一种新的未折叠蛋白反应 (UPR) 调节剂，可维持内质网 (ER) 中的稳态以响应错误折叠蛋白的压力。具体来说，HYPE 通过腺苷酸化 ER 伴侣 BiP/GRP78 来调节 UPR，它作为 UPR 激活的哨兵。维持 ER 稳态对于确定细胞命运至关重要，因此突出了 HYPE-BiP 相互作用的重要性。在这里，我们研究了决定 HYPE-BiP 相互作用的动力学和结构参数。通过测量 HYPE 在其野生型和 ATP 结合构象中的活化（腺苷酸化能力）和野生型（去腺苷酸化能力）形式的 BiP 的结合和动力学效率，我们确定 HYPE 显示出几乎相同的偏好野生型和 ATP 结合形式的 BiP 在体外和优先去 AMPylates 野生型的腺苷酸化 BiP。我们还表明，BiP 的 Thr366 与 Thr518 位点的 AMPylation 对其 ATP 酶活性有不同的影响，并且 HYPE 不会腺苷酸化 UPR 辅助蛋白，如 J 蛋白 ERdJ6。使用分子对接模型，我们解释了 HYPE 如何在体外使 Thr366 和 Thr518 位点腺苷酸化。虽然已经报道了 Thr366 和 Thr518 位点的 AMPylation 的生理作用，我们的分子对接模型支持 Thr518 作为结构上首选的修饰位点。这是对 HYPE-BiP 相互作用的第一次此类分析，并提供了对底物特异性和目标识别的重要见解。