Leptospira ClpP mutant variants in association with the ClpX, acyldepsipeptide, and the trigger factor displays...

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^E170D, LinClpP1^N172D, LinClpP2^IG_del, LinClpP2^S40AK41N, LinClpP2^Y62A) 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^S40AK41N and LinClpP2^Y62A) displayed gain-of-function (GOF) in peptidase activity. The ADEP1-bound heterocomplex (LinClpP1P2^S40AK41N and LinClpP1P2^Y62A) 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^S40AK41N and LinClpP1P2^Y62A) exhibited a 3-fold surge in peptidase activity. The deletion mutant (LinClpP2^IG_del) or its heterocomplex (LinClpP1P2^IG_del) displayed no activity. Similarly, the pure LinClpP1^E170D and LinClpP1^N172D could not cleave a model dipeptide. However, its heterocomplex (LinClpP1^E170DP2 and LinClpP1^N172DP2) showed 0.5-fold lower peptidase activity than the LinClpP1P2. Collectively, two mutants (LinClpP2^S40AK41N and LinClpP2^Y62A) have GOF and can degrade model dipeptide substrate without the aid of LinClpP1 isoform and thus provide new insights into unprecedented LinClpP activation.