Recombinant expression, biophysical and functional characterization of ClpS from

Abstract

Intracellular proteolysis is attracting more and more attention for its unique and important character in Mycobacterium tuberculosis (Mt). The ClpS protein from Mt (MtClpS) plays a critical role in intracellular proteolysis by recognizing N-end rule substrates, which makes it become a potential target for antibacterial drugs. However, the molecular mechanism of MtClpS recognizing N-end rule substrates remains unclear. Preparation of highly concentrated and pure MtClpS protein is a prerequisite for further structural and functional studies. In the present work, we tried several fusion tags and various expression conditions to maximize the production of MtClpS in Escherichia coli. We established an efficient approach for preparing the MtClpS protein with a high yield of 24.7 mg/l and a high purity of 98%. After buffer screening, we obtained a stable MtClpS protein sample concentrated at 0.63 mM in the presence of glycerol, l-Arginine, and l-Glutamate. Moreover, circular dichroism characterization indicated that the secondary structure of MtClpS consists of 38% α-helix and 24% β-sheet. The 2D 1H-15N HSQC nuclear magnetic resonance spectrum showed a good dispersion of resonance peaks with uniform intensity, indicating that the purified MtClpS protein was well folded and conformationally homogeneous. Isothermal titration calorimetry experiments revealed significant interactions of MtClpS with N-end rule peptides beginning with Leu, Tyr, Trp, or Phe. Furthermore, residues D34, D35, and H66 were confirmed as key residues for MtClpS recognizing the N-end rule peptide. The successful expression and biophysical characterization of MtClpS enabled us to gain insight into the molecular mechanism of MtClpS recognizing N-end rule substrates. The obtained stable and pure recombinant MtClpS will enable future inhibitor screening experiments.

ClpS directs degradation of primary N-end rule substrates inMycolicibacterium smegmatis

“…S3 ; Table 2 ). These results confirm that Msm ClpS can bind canonical N-end rule degrons, in agreement with studies on M. tuberculosis ClpS [25, 47], however the relatively weak interaction may not be sufficient to support robust degradation in cells. Sequence determinants beyond the leading amino acid may strengthen binding for some N-terminal sequences.…”

“…Recent studies point toward the existence of an N-end rule pathway in mycobacteria. Mycobacteria possess an ortholog of ClpS [46, 47] which has been shown to interact with ClpC1 [25]. Moreover, ClpS•ClpC1P1P2 degrades a model substrate bearing an N-terminal Phe in vitro [25].…”

Exploring a potential Achilles heel of Mycobacterium tuberculosis: defining the ClpC1 interactome

“…Consistent with the docking mechanism of E. coli ClpS to its cognate unfoldase ClpA [7,18], an interaction between the two proteins was observed. To validate this interaction, the team performed additional in vitro experiments using model substrates, confirming that Mtb ClpS was indeed responsible for the recognition of a model N‐degron substrate [19] and showed for the first time that Mtb ClpS is essential for the turnover of a model N‐degron substrate (FR‐li‐GFP) by ClpC1P1P2. Additionally, they showed that, similar to E. coli ClpS [6], Mtb ClpS also inhibited the in vitro turnover of a model C‐degron substrate, GFP‐ssrA and the auto‐degradation of its cognate unfoldase.…”