Activation Mechanism and Structural Assembly of the Mycobacterium tuberculosis ClpP1P2 Protease and Its Associated ATPases.

Abstract

Supramolecular assemblies are integral to cellular biochemical processes, relying on their dynamic nature to fulfill essential functions. The protease ClpP1P2, paired with ATPase partners ClpC1 or ClpX, is vital for the survival of Mycobacterium tuberculosis (Mtb). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides (e.g., Z-Leu-Leu) to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we identified novel activators that enabled the structural determination of the ClpC1P1P2 complex, providing insights into its assembly. Furthermore, we discovered that trehalose - a key metabolite and molecular crowding agent in Mtb, significantly enhances the activity of both ClpC1P1P2 and ClpXP1P2 complexes without the need for activating peptides. Analytical ultracentrifugation revealed that trehalose promotes the formation of these active complexes, mimicking intracellular conditions. These findings propose a new model of Clp system activation in vivo, offering promising avenues for therapeutic targeting in tuberculosis treatment. Significance StatementThe proteolytic complex formed by the essential proteins ClpP1 and ClpP2, along with their specific ATP-dependent activators ClpX and ClpC1, has emerged as a highly attractive target for anti-tuberculosis drug development. While previous studies have shown that ClpP1P2 can be activated in vitro by small peptide activators, its in vivo activation mechanism remains unclear. In this study, we identify novel activators and demonstrate that trehalose, a key metabolite in Mycobacterium tuberculosis, enhances ClpC1P1P2 and ClpXP1P2 activity without the need for activating peptides. These findings propose a new model for Clp system activation in Mycobacterium tuberculosis, advancing our understanding of its regulation and potential as a therapeutic target.