Abstract Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. ClpP protease is a tetradecameric complex that has emerged as a drug target against multiple pathogenic bacteria. During drug development efforts, the activation of different ClpPs by inhibitors was independently reported, but so far, no rationale for inhibitor-induced activation has been proposed. Using an integrated approach that included X-ray crystallography, solid-and solution-state NMR, MD simulations and ITC we show that the proteasome-inhibitor bortezomib binds to the ClpP active site serine mimicking a peptide substrate and induces the concerted allosteric activation of the complex. The bortezomib activated conformation also displays a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association as well as protein processive degradation.