Structural basis for TetM-mediated tetracycline resistance

Abstract

Ribosome protection proteins (RPPs) confer tetracycline resistance by binding to the ribosome and chasing the drug from its binding site. The current model for the mechanism of action of RPPs proposes that drug release is indirect and achieved via conformational changes within the drug-binding site induced upon binding of the RPP to the ribosome. Here we report a cryo-EM structure of the RPP TetM in complex with the 70S ribosome at 7.2-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM and the decoding center of the small subunit. Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance. A model is presented whereby TetM directly dislodges tetracycline to confer resistance.

Fig. 1.

Cryo-EM reconstruction of a TetM•70S complex. (A) Final map of the TetM•70S complex with TetM (orange), 30S (yellow), and 50S (gray). (b, body; CP, central protuberance; h, head; sp, spur). (B) Schematic color code of the domain structure of EF-G and TetM (domain I, G′ subdomain, II, III, IV, V, and CTE are shown in green, blue, red, yellow, pink, pale blue, and orange, respectively), with fit of the homology model for TetM into the extracted cryo-EM density (gray mesh). (C) Relative binding position of domain IV of TetM (orange) compared with mRNA (tan), A-tRNA (green), and P-tRNA (blue). (D) Relative positions of domain V of TetM (orange) and EF-G (22) (blue) with their respective stalk base regions (H43/H44 and L11-NTD) colored pale blue and yellow, respectively. Arrows in D indicate the shift in the position of the stalk base between TetM and EF-G.

Fig. 2.

Localization and interaction of the CTE of TetM. (A) Density (gray mesh) of domain IV of TetM fitted with homology model (colors are as in Fig. 1B). The arrow indicates the site where the homology with EF-G ends, yet additional density is observed extending from domain V toward domain IV (asterisk). (B) Same as A but with extended CTE (orange), modeled based on (C) PSIPRED secondary structure prediction, with sequence (Seq.), prediction (Pred.), and probability (Prob.) as indicated. Underlined residues were deleted to create TetM-ΔCTE. (D and E) Interaction of the CTE of TetM (orange) with H69 (blue) of the 23S rRNA and h44 of the 16S rRNA (pale blue), modeled with A1492 and A1493 flipped out of h44 [Protein Data Bank ID 2XQD (25)].

Fig. 3.

Interaction of domain IV of TetM at the tetracycline binding site. (A) Overview of cryo-EM density (gray mesh) with the 30S subunit (blue) and TetM model (orange). (b, body; h, head; pl, platform; sp, spur.) (B) Schematic representation of secondary structure elements for TetM-domain IV, with α-helices, β-sheets, and loops I to III indicated as well as the CTE. (C) Cryo-EM density (gray mesh) reveals interaction of loop II (spheres for Cα of Ser465–Leu466–Gly467) with the backbone of h34 (C1208–C1209), and loop III (spheres for Cα of Tyr507–Ser508–Pro509–Val510) with C1054 of the 16S rRNA. (D) Same view as in C for SecM-stalled ribosome nascent chain complex (SecM-RNC) with an empty A-site (EMD-1829) (26). (E and F) Electron density map (gray mesh) of (E) TetM•70S showing lack of density for Tgc in comparison with (F) the nonrotated 70S map without TetM from sorting (SI Appendix, Fig. S2) that reveals density for Tgc.

Fig. 4.

The role of loop III residues in TetM in tetracycline resistance. (A) Cryo-EM density of TetM•70S map (gray mesh) reveals relative proximity of loop III residues Tyr506–Tyr507–Ser508–Pro509–Val510 (spheres for Cα) to C1054 of the 16S rRNA and the primary tetracycline binding site (Tet) (2). (B) Growth curves of WT E. coli strain BL21 (−TetM, black) in the presence of increasing concentrations of tetracycline (0–128 μg/mL) compared with the WT strain harboring a plasmid encoding WT E. faecalis TetM (+TetM, red), C-terminally truncated TetM (TetM-ΔCTE, pink), TetM-YSP/AAA, (orange), TetM-SPV/AAA (purple), TetM-YY/AA (blue), TetM-Y506A (yellow), and TetM-Y507A (green). (C) Cryo-EM density of TetM•70S map (gray mesh) with comparison of relative positions of C1054 and U1196 of 16S rRNA from TetM•70S model [based on EF-Tu•70S structure (25), blue] and tetracycline-30S structure (pale red) (2). (D) Conformation of C1054 and U1196 of 16S rRNA from EF-Tu•70S structure (blue) (25), with A-tRNA (green) and mRNA (yellow) compared with tetracycline-30S structure (pale red) (2).

Fig. 5.

A model for the mechanism of TetM-mediated tetracycline resistance. (A) Binding of tetracycline (Tet, red) and interaction with C1054 (light blue) within h34 of the 30S subunit prevents delivery of the ternary complex consisting of EF-Tu•GTP (purple) and aa-tRNA (green) to the ribosomal A-site. (B) The tetracycline-bound ribosome is recognized and bound by TetM•GTP (orange; CTE and loop III are shown in black with the important residues Y506 and Y507 as yellow spheres). TetM binding induces nucleotides A1492 and A1493 to flip out of h44 and interact with the CTE of TetM. Loop III of TetM dislodges tetracycline from the ribosome (red arrow) and prevents rebinding by changing the conformation of C1054 (changed conformation in green). The stalk base (SB) adopts a position similar to when EF-Tu is bound, and, additionally L7 interacts with the G′ subdomain of TetM to catalyze Pi release and TetM dissociation. (C) Despite the dissociation of TetM•GDP, the conformational changes in the ribosome remain, preventing rebinding of tetracycline and promoting binding of ternary complex EF-Tu•GTP•aa-tRNA so translation can continue.