Abstract 2374 Structural Insights into the Activation Mechanism of Human ClpXP

The human ClpXP complex (hClpXP), a member of to the ATPases associated with diverse cellular activities (AAA+) protease family, consists of the ATPase ClpX and the protease ClpP. This complex plays a crucial role in mitochondrial protein quality control by selectively degrading misfolded or damaged proteins using the energy from ATP hydrolysis. Beyond its involvement in protein quality control, hClpXP is intricately linked to various cellular processes such as oxidative phosphorylation and heme biosynthesis. Despite the extensive characterization of prokaryotic ClpXP over the past two decades, our understanding of the regulatory functions, substrate recognition, and degradation mechanisms of hClpXP remains limited. In this study, we present cryo-electron microscopy structures of hClpP and hClpXP in a substrate-translocating state. We show that hClpP exists as an inactive heptamer independently and forms an active tetradecamer upon binding with hexameric hClpX. A detailed comparison of hClpP alone and hClpX-bound hClpP structures highlights substantial conformational changes during the transition from an inactive to an active state. Notably, hClpP features a unique C-terminal extension, and we unveil its regulatory role in hClpXP interaction by physically obstructing the hClpX binding pockets on hClpP. The removal of this extension in hClpP resulted in a tighter binding to hClpX and a faster rate of substrate degradation. Altogether, these structures and biochemical data enhance our understanding of the complex mechanism of action of hClpXP, offering a valuable framework for future therapeutic applications. Wenqian Chen is supported by a National Science Foundation predoctoral fellowship.