Fig. 1: The FRB-FKBP dimerizable domains can be used to induce proximity in mycobacteria. | Nature Communications

Fig. 1: The FRB-FKBP dimerizable domains can be used to induce proximity in mycobacteria.

From: Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy

Fig. 1

a The concept of bacterial targeted protein degradation (TPD), in which an essential target protein is delivered to the ClpC1P1P2 proteolytic complex by a heterobifunctional molecule. b Schematic of our chemical-genetic approach to induced proximity, in which fusions to the FRB and FKBP domains are dimerized with the addition of rapamycin. c Luminescence in live cells expressing split NanoLuc (Nluc) fragments with and without fusion to FRB and FKBP measured in relative light units (RLU). Density matched log phase cells incubated with the luciferase substrate furimazine and a range of rapamycin concentrations at 37 °C for 10 min. d Crystal structure of the stabilized mutant MtbClpC1 hexamer (teal, PDB: 8A8U) with highlighted monomer (translucent green) and three visible N-terminal domains (NTDs) which cannot be assigned to specific protomers due to invisibility of the linker region (grayscale, PDB: 6PBS). ClpC1 N- and C-terminal residues highlighted in green and magenta, respectively. e Fluorescence of eGFP-ssrA measuring in vitro protease activity of ClpC1P1P2 complex with WT MtbClpC1 (left) or FRB-ClpC1 and ClpC1-FRB (right). Purified MtbClp proteins incubated with eGFP-ssrA substrate at 37 °C. f (top) Schematic of the clpC1 L5 allele swap, in which a second copy of clpC1 is integrated at the L5 phage attB site. This enables recombineering-mediated knockout of chromosomal (Chr::) clpC1, followed by an L5 integrase-mediated swap for the clpC1-frb allele and an alternative resistance marker. (bottom) Quantification of the clpC1-frb swap; true swaps carry only the second resistance marker, whereas double integrants carry both (and both clpC1 alleles). For (c), data are mean ± s.d. of three technical replicates and are representative of two independent experiments; e, data are individually plotted technical replicate measurements, normalized to time = 0 h, and are representative of two independent experiments. a, b Created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided with this paper.

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