Leptospira ClpP mutant variants in association with the ClpX, acyldepsipeptide, and the trigger factor displays unprecedented gain-of-function.

The functionally active ClpP (LinClpP) of Leptospira interrogans is composed of two different isoforms (LinClpP1 and LinClpP2). In this study, five mutants of LinClpP (LinClpP1, LinClpP1, LinClpP2, LinClpP2, LinClpP2) targeting its critical hotspot residues were generated. The functional activity of pure LinClpP mutant variants or its heterocomplex and its effect when associated with a chaperone (LinClpX)/antibiotic acyldepsipeptide (ADEP1)/trigger factor (LinTF) was examined. The two mutants (LinClpP2 and LinClpP2) displayed gain-of-function (GOF) in peptidase activity. The ADEP1-bound heterocomplex (LinClpP1P2 and LinClpP1P2) measured 1.7 and 1.5-fold higher protease activity than ADEP-bound LinClpP1P2. The dynamic light scattering analysis of ADEP1-bound GOF mutants displayed increased hydrodynamic diameter. In the presence of LinTF, the heterocomplex (LinClpP1P2 and LinClpP1P2) exhibited a 3-fold surge in peptidase activity. The deletion mutant (LinClpP2) or its heterocomplex (LinClpP1P2) displayed no activity. Similarly, the pure LinClpP1 and LinClpP1 could not cleave a model dipeptide. However, its heterocomplex (LinClpP1P2 and LinClpP1P2) showed 0.5-fold lower peptidase activity than the LinClpP1P2. Collectively, two mutants (LinClpP2 and LinClpP2) have GOF and can degrade model dipeptide substrate without the aid of LinClpP1 isoform and thus provide new insights into unprecedented LinClpP activation.