Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX
2019-07-31T08:14:00Z (GMT) by
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 active rClpP isoform mixture fractionates as a tetradecamer. The distinct and unprecedented oligomeric form of rClpP1 was also evident through native-gel and dynamic light scattering. Moreover, the rClpP isoform mixture formed after the site-directed mutation of either or both the isoforms at one of the catalytic triad residues (Ser 98/97 to Ala 98/97) resulted in the complete loss of protease activity. The rClpP isoform mixture gets stimulated to degrade the casein substrate in the presence of rClpX and in an energy-dependent manner. On the contrary, pure rClpP1 or the rClpP2 isoform in association with rClpX are incapable of forming operative protease. The reported finding suggests that in Leptospira, the enzymatic activity of the rClpP protease complex in the presence or absence of cochaperone is performed solely by the tetradecamer structure which is hypothesized to be composed of 2-stacked ClpP heptameric rings, wherein each ring is a homo-oligomer of ClpP1 and ClpP2 subunits. Understanding the activities and regulation principle of multi-isoforms of ClpP in pathogenic bacteria may aid in intervening disease outcomes particularly to the co-evolving antibiotic resistance strains.