Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2

Abstract

Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin.

Figure 1

(A) The amino acid sequence and post-translational modifications of lassomycin. Blue numbering indicates the positions of residues 1, 8 and 16. (B) The backbone structure of lassomycin. The N- and C-termini are labeled. (C) The structure of lassomycin with its side chains shown.

Figure 2

The putative lassomycin biosynthetic operon.

Figure 3

Time-dependent killing of M. tuberculosis mc²6020 by lassomycin. All drugs were administered at 10 ×MIC. Each point represents the average of three biological replicates. Rifampicin (red circles), lassomycin (green triangles), or untreated (black squares) of exponential (A) and stationary (B) M. tuberculosis. Dashed blue line indicates the limit of detection. The error bars represent standard deviation.

Figure 4

Sequences of the two N-terminal repeat regions of ClpC1 mutants resistant to lassomycin. Amino acid changes are indicated in red.

Figure 5. Lassomycin effect on ClpC1 ATPase activity and protein degradation by ClpC1P1P2 complex

A) Lassomycin stimulates ATPase activity of ClpC1. 32 nM of pure ClpC1 were mixed with 100 μl of the assay buffer (50 mM TrisHCl pH 7.8; 100 mM KCl; 10% glycerol; 1 mM phosphoenolpyruvate; 1 mM NADH; 2 units of pyruvate kinase/lactic dehydrogenase (Sigma); 4 mM MgCl2 and 1 mM ATP) and the ATPase activity of ClpC1 was followed by measuring the coupled oxidation of NADH to NAD⁺ spectrometrically at 340 nm. Similar results were obtained when the ATPase activity was measured with the Malachite Green method (Geladopoulos et al., 1991). The rate of ATPase activity in the absence of lassomycin was taken as 100%. The apparent K_d and Hill coefficient for lassomycin activation of ClpC1 ATPase were determined using curve fitting with classic Hill-kinetic through a Scaled Levenberg-Marquardt algorithm; tolerance 0.0001. B) ClpC1 does not activate degradation of casein by ClpP1P2 in the presence of lassomycin. ClpP1P2 (100 nM) and ClpC1 (100 nM) were mixed in 80µlofreaction buffer containing 50 mM potassium phosphate (pH 7.6), 100 mM KCl, 8 mM MgCl₂, 5% glycerol, 2 mM ATP, 5 mM Z-Leu-Leu. Enzymatic activity was measured fluorometrically using FITC-casein as a substrate in the presence or absence of 10 µM of lassomycin. The rate of degradation of ClpP1P2 was taken as 100%. C) Lassomycin does not interfere with casein binding to ClpC1. ATPase activity of ClpC1 (32nM) was measured as in Fig. 5A in the presence or absence of casein (10 µM) and lassomycin (1 µM). The activity of ClpC1 alone (control) was taken as 100%. D) Stimulation of ClpC1 ATPase activity by lassomycin is highly specific. The activities of purified ATPases from bacteria (M. tuberculosis ClpX; E.coli ClpA, ClpB and GroEL), archaea (PAN) and mouse (26S proteasome) were measured in the presence and absence of lassomycin (10 μM). ATPase activity of each ATPase in the absence of lassomycin was taken as 100%.

Figure 6. Images of a docking result and structure evaluations

(A) Lassomycin (purple) binds at the mutation sites in the M. tuberculosis ClpC1 N-domain (green; C-terminus in red). Mutation sites are labeled and shown in orange. (B) Transparent overlapping surface map shows electrostatics, making positively charged regions (blue) and negatively charged regions (red) visible. Protein backbone in orange, mutant residues in green. (C) The temperature-factor value of the top loop of the ClpC1N-terminus crystal structure 3WDB indicates a very flexible part (red) of the molecule. The color changes from very flexible in red to very rigid in blue. (D) Mutant ClpC1 model variants yield fewer binding positions for the region of the lassomycin-resistant mutations than the wild-type model (WT). Docking was performed assuming a flexible (black) or rigid (shaded) backbone of lassomycin.