Recombinant expression, biophysical and functional characterization of ClpS from Mycobacterium tuberculosis.

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% ß-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.

Crystal structure of the N domain of Lon protease from Mycobacterium avium complex.

Lon protease is evolutionarily conserved in prokaryotes and eukaryotic organelles. The primary function of Lon is to selectively degrade abnormal and certain regulatory proteins to maintain the homeostasis in vivo. Lon mainly consists of three functional domains and the N-terminal domain is required for the substrate selection and recognition. However, the precise contribution of the N-terminal domain remains elusive. Here, we determined the crystal structure of the N-terminal 192-residue construct of Lon protease from Mycobacterium avium complex at 2.4 å resolution，and measured NMR-relaxation parameters of backbones. This structure consists of two subdomains, the ß-strand rich N-terminal subdomain and the five-helix bundle of C-terminal subdomain, connected by a flexible linker，and is similar to the overall structure of the N domain of Escherichia coli Lon even though their sequence identity is only 26%. The obtained NMR-relaxation parameters reveal two stabilized loops involved in the structural packing of the compact N domain and a turn structure formation. The performed homology comparison suggests that structural and sequence variations in the N domain may be closely related to the substrate selectivity of Lon variants. Our results provide the structure and dynamics characterization of a new Lon N domain, and will help to define the precise contribution of the Lon N-terminal domain to the substrate recognition.