Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX

Leptospira interrogans genome is predicted to encode multiple isoforms of caseinolytic proteases (ClpP1 and ClpP2). The ClpP proteins with the aid of its ATPase chaperone are known to be involved in establishing cellular proteostasis and have emerged as a target for developing new antibiotics. We report the molecular characterization of recombinant ClpP1 (rClpP1) and rClpP2 of Leptospira along with its ATPase chaperone rClpX. The two isoforms of rClpPs when coupled together in an equivalent concentration exhibit optimum activity on small fluorogenic peptide substrates, whereas the pure rClpP isoforms are enzymatically inactive. Isothermal titration calorimetry analysis suggests that the two rClpP isoforms bind each other moderately in a 1:1 stoichiometry with a dissociation constant of 2.02 ± 0.1 μM at 37 °C and is thermodynamically favored. Size exclusion chromatography fractionates the majority of pure rClpP1 at ≥308 kDa (14–21-mer) and the pure rClpP2 at 308 kDa (tetradecamer), whereas the functionally...