The tetracycline resistance protein Tet(○) perturbs the conformation of the ribosomal decoding centre

Abstract

SummaryTet(O) is an elongation factor-like protein found in clinical isolates of Campylobacter jejuni that confers resistance to the protein-synthesis inhibitor tetracycline. Tet(O) interacts with the 70S ribosome and promotes the release of bound tetracycline, however, as shown here, it does not form the same functional interaction with the 30S subunit. Chemical probing demonstrates that Tet(O) changes the reactivity of the 16S rRNA to dimethyl sulphate (DMS). These changes cluster within the decoding site, where C1214 is protected and A1408 is enhanced to DMS reactivity. C1214 is close to, but does not overlap, the primary tetracycline-binding site, whereas A1408 is in a region distinct from the Tet(O) binding site visualized by cryo-EM, indicating that Tet(O) induces long-range rearrangements that may mediate tetracycline resistance. Tetracycline enhances C1054 to DMS modification but this enhancement is inhibited in the presence of Tet(O) unlike the tetracycline-dependent protection of A892 which is unaffected by Tet(O). C1054 is part of the primary binding site of tetracycline and A892 is part of the secondary binding site. Therefore, the results for the first time demonstrate that the primary tetracycline binding site is correlated with tetracycline's inhibitory effect on protein synthesis.

Structural basis for TetM-mediated tetracycline resistance

“…3D). This additional density in the TetM•70S map fuses directly with C1054 of the 16S rRNA, a component of the primary tetracycline binding site (2, 3), and is consistent with the protection of C1054 from DMS modification observed upon TetO binding to the ribosome (15). Although domain IV of TetO was separated by 6 Å from the tetracycline binding site in the previous cryo-EM reconstruction of the TetO•70S complex (14), we believe that this arises from the limited resolution of the TetO•70S complex: Filtering of the TetM•70S cryo-EM map to a similar resolution as the TetO•70S complex also leads to loss of density for loop III of domain IV of TetM (SI Appendix, Fig.…”

“…2E). Binding of TetO to the ribosome leads to an enhancement in the chemical reactivity of A1408 of the 16S rRNA to DMS modification (15). Consistently, the stacked conformation of A1493 would protect A1408 from modification (SI Appendix, Fig.…”

“…Collectively, these results suggest that binding of both TetM (and TetO) to the ribosome leads to the flipping out of A1492 and A1493-a conformation that is stabilized via interaction with the CTE of TetM. The enhancement of A1408 is also observed when TetO is bound with GTP rather than GDPNP (15), suggesting that the flipped-out conformation of A1492 and A1493 remains after the RPP has left the ribosome.…”

“…In contrast to EF-G, the tip of domain IV of TetO does not significantly overlap with the A-tRNA, but rather interacts with h34 adjacent to the tetracycline binding site (14). Binding of TetO to the ribosome leads to protection of C1214 and to a lesser extent C1054 within h34 from chemical modification, whereas the reactivity of A1408 in h44 is enhanced (15). As TetO is not observed to directly interact with C1054 or A1408 (14), TetO was suggested to chase tetracycline from the ribosome indirectly via inducing local disturbances within h34 (9,14,15).…”

“…Binding of TetO to the ribosome leads to protection of C1214 and to a lesser extent C1054 within h34 from chemical modification, whereas the reactivity of A1408 in h44 is enhanced (15). As TetO is not observed to directly interact with C1054 or A1408 (14), TetO was suggested to chase tetracycline from the ribosome indirectly via inducing local disturbances within h34 (9,14,15). Moreover, the conformational changes were proposed to persist after TetO has dissociated from the ribosome, preventing rebinding of tetracycline as well as stimulating delivery of the ternary complex (9,16).…”