Fig. 3: Prc and DegP target apoNDM-1 at specific sites.

a 2D ¹H-¹⁵N HSQC spectrum of the supernatant of E. coli cells overexpressing ¹⁵N/¹³C-isotopically enriched NDM-1 and non-enriched Prc/DegP after DPA exposure and incubation for 4 h at 20 °C. Colored circles denote the newly generated C-termini and the amino acid type: Cyan (alanine), light brown (valine), burgundy (isoleucine) and green (threonine). The assignment of newly generated C-termini and amino acid type was determined by chemical shifts analysis of CO, Cα and Cβ of the corresponding resonance (b, c). d 2D ¹H-¹⁵N HSQC spectra in the backbone carboxylate region of apoNDM-1 processed by Prc and DegP (black) and by Prc (ΔdegP background, gray) or DegP (Δprc background, green). Blue arrows indicate NDM-1 NMR signals with chemical shifts displacements when one or both proteases were present. Inset show the spectral region of threonine C-termini. NMR spectra were acquired at 20 °C. e Pie chart of apoNDM-1 C-termini generated in the bacterial periplasm by Prc and DegP (left), Prc or DegP (right, first row) and in vitro by Prc or DegP (right, second row). f Representative immunofluorescence of NDM-1 (magenta) processing by Prc or DegP (green) in E. coli cells (N = 3, n = 10 per treatment). g Analysis of apoNDM-1 degradation by Prc and DegP, Prc or DegP in live E. coli cells at 20 °C. ApoNDM-1 levels were measured by Western blot. The data represents mean ± SD. (N = 4). Significant difference *P < 0.05 and ****P < 0.0001, Dunnet, two-way ANOVA. Exact, mean ± SD values are enclosed in the source data file.