Mechanism of allosteric activation in human mitochondrial ClpP protease
Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement. Significance statementHuman ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.