Trigger Factor in Association with the ClpP1P2 Heterocomplex of Leptospira Promotes Protease/Peptidase Activity

Abstract

The genomic analysis of Leptospira reveals a trigger factor (TF) encoding gene (tig) to be colocalized along with the clpP1 and clpX. The TF is a crouching dragon-like protein known to be a ribosomeassociated chaperone that is involved in cotranslational protein folding in bacteria in an ATP-independent mode. In Leptospira, tig is localized upstream of the clpP1 with a short (4 bp) overlap. In the present study, we document the distinctive role of Leptospira TF (LinTF) in the caseinolytic protease (ClpP) system. The recombinant LinTF (rLinTF) was found to improve the peptidase or protease activity of the ClpP1P2 heterocomplex and ClpXP1P2 complex, respectively, on model substrates. In addition, on supplementation of rLinTF to rClpP1P2 bound to its physiological ATPase chaperone ClpX or the antibiotic analogue acyldepsipeptide (ADEP), an augmentation in the activity of ClpP1P2 was observed. These studies underscore the novel role of LinTF in aiding the caseinolytic protease activity of Leptospira. Supplementation of rLinTF to a peptidase assay of rClpP1P2 conditionally in the presence of a salt (sodium citrate) with high Hofmeister strength led us to speculate that rLinTF may have a role in the assembly of multimeric proteins. The deletion of one of the arms (arm-2) of the LinTF structure from the carboxy terminal domain indicated a reduction in its capacity to stimulate rClpP1P2 activity. Thus, the Cterminal domain of LinTF may have a role in the assembly of multimeric ClpP protein, leading to enhancement of ClpP activity.

Reprogramming of the Caseinolytic Protease by ADEP Antibiotics: Molecular Mechanism, Cellular Consequences, Therapeutic Potential

“…Peptide array mapping, mutagenesis and NMR analyses jointly support an interaction model which involves all three domains of TF and the AAA+ plus zinc-binding-domain of ClpX, establishing TF as a new adapter for ClpX (Rizzolo et al, 2021). However, TF also impacts the activity of the ClpP core when ClpX is absent (Choudhury et al, 2021). In Leptospira, TF stimulated the peptidase activity of ClpP1P2 and also casein degradation by the ADEP-activated Leptospira ClpP1P2 core (Choudhury et al, 2021).…”

“…However, TF also impacts the activity of the ClpP core when ClpX is absent (Choudhury et al, 2021). In Leptospira, TF stimulated the peptidase activity of ClpP1P2 and also casein degradation by the ADEP-activated Leptospira ClpP1P2 core (Choudhury et al, 2021). As demonstrated by these examples, ADEP is also increasingly used as a research tool to better characterize new ClpP homologs.…”

“…In Leptospira , trigger factor (TF) is chromosomally colocalized with ClpP and ClpX, and the same applies to E. coli and many other organisms. This observation led two research teams to test a potential functional connection ( Choudhury et al, 2021 ; Rizzolo et al, 2021 ). The addition of TF to Leptospira ClpXP stimulated casein degradation by the protease ( Choudhury et al, 2021 ).…”

“…This observation led two research teams to test a potential functional connection ( Choudhury et al, 2021 ; Rizzolo et al, 2021 ). The addition of TF to Leptospira ClpXP stimulated casein degradation by the protease ( Choudhury et al, 2021 ). In the E. coli study, proteolytic activation of ClpXP by Tig was demonstrated for a broader range of protein substrates in vitro , and from pulse-chase experiments in E. coli cells it was estimated that about 2% of newly synthesized proteins are degraded in a TF-dependent manner ( Rizzolo et al, 2021 ).…”