Elucidating the structural basis of ClpP activation and dynamics in <i>Mycobacterium tuberculosis</i>

The rise of drug-resistant <i>Mycobacterium tuberculosis</i> (Mtb) strains has driven the search for novel therapeutic targets beyond conventional anti-tubercular agents. One such promising target is the ClpP protease complex, composed of ClpP1 and ClpP2 subunits, which is essential for proteostasis and bacterial survival under stress. This study explores the molecular dynamics (MD) and activation mechanism of Mtb ClpP subunits by <i>N</i>-[(benzyloxy)carbonyl]-<i>L</i>-isoleucyl-L-leucine (ZIL), an N-blocked dipeptide activator. MD simulations (200-1000 ns) were used to analyze structural stability, ligand interactions, and domain dynamics of both subunits in active and inactive states. ZIL-bound simulations showed that ClpP1 and ClpP2 maintained structural integrity, with conserved ligand-proximal residues forming stable interactions, although ClpP2 exhibited more variable polar contacts. In contrast, ligand-free simulations (500 ns) revealed significant instability, particularly in the handle domain and S1 binding pocket, underscoring the stabilizing role of ZIL. A 1000 ns simulation, with ZIL placed away from its known binding site on inactive ClpP1, showed that the ligand approached its target site and triggered a conformational shift in the handle domain, an early allosteric response, even though it did not fully dock as observed in the crystal structure. Notably, the residues in proximity to ZIL were associated with the observed structural changes in the simulations. The resulting MD trajectories provide a continuous, atomic-level view of ligand-induced dynamics and early activation events. Conducted without prior mechanistic assumptions, this unbiased simulation highlights the potential of targeting allosteric activation mechanisms and offers valuable insight into the rational design of ClpP-based therapeutics against drug-resistant Mtb.