How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.

AAA +分子构象如何以ATP依赖性方式重塑特定的靶蛋白尚不清楚。在这里，我们研究了AAA +蛋白Rubisco激活酶（Rca）在光合作用酶Rubisco的代谢修复中的机制，该光合作用是8个大（RbcL）和8个小（RbcS）亚基的复合物，其中包含8个催化位点。Rubisco易于受到紧密结合的糖磷酸酯的抑制，而Rca催化磷酸的去除。我们设计了一个稳定的Rca六聚环，并分析了其与Rubisco的功能相互作用。氢/氘交换和化学交联表明，Rca具有显着的选择性，使Rubisco活性位点的元素不稳定。低温电子显微镜显示，Rca一次在一个活性位点停靠在Rubisco上，将RbcL的C端链定位，它可以稳定催化中心，以便进入Rca六聚体孔。Rca的拉力经过微调，可以避免整体不稳定，并可以进行精确的酶修复。