AAA(与多种细胞活动相关的ATP酶)的功能丧失与疾病有关,激活这些蛋白质的小分子可以成为探测机制和测试治疗假设的有力工具。与可以结合单一构象状态来阻断酶功能的化学抑制剂不同,活化剂结合必须允许机械化学所需的不同构象状态。然而,我们不知道AAA蛋白是如何被小分子激活的。这里,我们专注于含valosin的蛋白质(VCP)/p97,这是一种AAA复折酶,其功能丧失与基于蛋白质聚集的疾病有关,确定化学活化剂的可药用部位。我们鉴定了VCPATP酶激活剂1(VAA1),一种剂量依赖性地刺激VCPATP酶活性高达3倍的化合物。我们的低温EM研究得出了处于apo和ADP结合状态的VCP的结构(范围从〜2.9到3.7µ分辨率),并揭示了VAA1在两种状态下都结合了C末端附近的变构口袋。VAA1结合位点中的工程化突变赋予对VAA1的抗性,此外,调节VCP活性。可以占据VAA1结合位点的VCPC末端尾部中的苯丙氨酸残基的突变也刺激ATPase活性,这表明VAA1通过模仿这种相互作用来发挥作用。一起,我们的发现揭示了一个可药物化的变构位点和一个可通过小分子模拟调节的酶调节机制.
The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.