Structural basis for TetM-mediated tetracycline resistance

Domain IV of TetM directly interacts with the tetracycline binding site.

“Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance.”

• Also stated in: § Domain IV of TetM Directly Encroaches Upon the Tetracycline Binding · plain

  “Domain IV of TetM Directly Encroaches Upon the Tetracycline Binding”

Domain IV of TetM interacts with the cleft between the head and body of the small subunit.

“Domain IV of TetM interacts with the cleft between the head and body of the small subunit (Fig. 3A).”

Density connects the distal end of helix-αA of the TetM C-terminal extension with helix 44 of the 16S rRNA, in close proximity to A1492 and A1493.

“Additionally, density is seen connecting the distal end of helix-αA of the TetM-CTE with helix 44 of the 16S rRNA, in close proximity to A1492 and A1493 (Fig. 2E).”

Loop II of TetM interacts with the proximal end of h34 of the 16S rRNA, with residues Ser465-Leu466-Gly467 coming into close proximity with the backbone of C1209 and the nucleobase of C1214.

“Loop II between β4 4 and αA 4 interacts with the proximal end of h34 of the 16S rRNA, with residues Ser465-Leu466-Gly467 ( 465 SLG 467 ) coming into close proximity with the backbone of C1209 and the nucleobase of C1214 (Fig. 3C).”

Additional density in the TetM•70S map fuses directly with C1054 of the 16S rRNA, a component of the primary tetracycline binding site.

“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).”

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The overall orientation of TetM on the ribosome is similar to that observed previously for TetO.

“The overall orientation of TetM on the ribosome is similar to that observed previously for TetO (14), although no direct comparison can be made because the TetO map was not deposited in a public database.”

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Residues critical for conferring tetracycline resistance were identified.

“Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance.”

The C-terminal extension of TetM was modeled as a short 11-amino-acid α-helix (residues 627-637) connected to domain V by a flexible unstructured loop.

“Therefore, the CTE of TetM was modeled as a short 11-aa α-helix (residues 627-637) connected to domain V by a flexible unstructured loop (Fig. 2B), in accordance with secondary structure predictions (Fig. 2C).”

TetM significantly overlaps with the anticodon stem-loop of the A-tRNA.

“TetM significantly overlaps with the anticodon stem-loop of the A-tRNA (Fig. 1C), as observed for EF-G bound to a posttranslocation state ribosome (22).”

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No density for tigecycline is observable in the TetM•70S map although the complex was formed in the presence of 10 μM tigecycline.

“Although the TetM•70S complex was formed in the presence of 10 μM Tgc, no density for the drug is observable in the TetM•70S map (Fig. 3E).”

Three loops protrude from one end of domain IV of TetM, referred to as loops I, II, and III.

“Three loops protrude from one end of domain IV of TetM, hereafter referred to as loops I, II, and III (Fig. 3B).”

A cryo-EM structure of the ribosome protection protein TetM in complex with the 70S ribosome was determined at 7.2-Å resolution.

“Here we report a cryo-EM structure of the RPP TetM in complex with the 70S ribosome at 7.2-Å resolution.”

• Also stated in: § Structural basis for TetM-mediated tetracycline resistance · plain

  “we have determined a cryo-EM structure of the RPP TetM bound to the 70S ribosome at 7.2-Å resolution.”

Tyrosine 507 of TetM comes into close proximity of C1054 in the TetM•70S model.

“The equivalent residue to H583 of E. coli EF-G in TetM is tyrosine 507 (Y507; SI Appendix, Fig. S1), which, in the TetM•70S model, comes into close proximity of C1054 (Fig. 4A).”

No single alanine substitution within loop III of TetM exhibited a significant effect on the ability of TetM to confer tetracycline resistance.

“Surprisingly, no single alanine substitution within loop III of TetM exhibited a significant effect on the ability of TetM to confer tetracycline resistance (shown for Y506A and Y507A in Fig. 4B)”

Loop III of TetM contains a number of residues that are highly conserved in ribosome protection proteins, in particular the 508SPVS511 motif that directly follows Y507.

“Moreover, loop III contains a number of residues that are highly conserved in RPPs, in particular the 508 SPVS 511 motif that directly follows Y507 (SI Appendix, Fig. S1).”

Pelleting assays confirmed that binding of TetM to the 70S ribosome was observed in the presence of tigecycline.

“Pelleting assays confirmed that binding of TetM to the 70S ribosome was observed in the presence of Tgc, suggesting that analysis of this complex by cryo-EM may enable TetM and Tgc to be visualized simultaneously on the same ribosome.”

No additional density for tigecycline is observed within any of the secondary tetracycline binding sites in the TetM•70S map.

“In contrast to tetracycline, which has multiple binding sites on the ribosome (2, 3), a careful examination of the TetM•70S map reveals no additional density for Tgc within any of the sec- ondary tetracycline binding sites (SI Appendix, Fig. S10 I-M).”

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Density for tigecycline is clearly present in the nonrotated and rotated 70S ribosomes where TetM is not bound.

“Density for Tgc is, however, clearly present in the nonrotated (Fig. 3F) and rotated (SI Appendix, Fig. S10 A-C) 70S ribosomes where TetM is not bound.”

Overexpression of E. faecalis TetM raises the tetracycline MIC50 by 14-fold to approximately 10 μg/mL.

“whereas, as expected, overexpression of E. faecalis TetM (Fig. 4B, red circles) raises the MIC 50 by 14-fold to ∼10 μg/mL (Fig. 4B).”

Binding of TetM to the ribosome induces A1492 and A1493 to adopt a flipped-out conformation analogous to that observed during decoding of the mRNA by EF-Tu•GTP•aa-tRNA.

“Moreover, binding of TetM to the ribosome induces A1492 and A1493 to adopt a flipped-out conformation (Fig. 2 C and D), analogous to that observed during decoding of the mRNA by EF-Tu•GTP•aa-tRNA (25) (Fig. 4D).”

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E. faecalis TetM is comprised of five domains, labeled I through V.

“Like EF-G, E. faecalis TetM is comprised of five domains, labeled I through V (Fig. 1B).”

TetM induces a conformation in the stalk base analogous to that observed in the EF-Tu•70S structure.

“Finally, we observe that TetM also induces a conformation in the stalk base, analogous to that observed in the EF-Tu•70S structure (25).”

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In the absence of TetM protein, the wild-type E. coli strain BL21 is sensitive to tetracycline with a minimal inhibitory concentration (MIC50) of approximately 0.6 μg/mL.

“In the absence of TetM protein, the WT E. coli strain (Fig. 4B, black squares) is sensitive to tetracycline with minimal inhibition concentration (MIC 50 ) of ∼0.6 μg/mL”

TetM binds preferentially to the nonrotated 70S ribosome state.

“The absence of a subpopulation of TetM bound to rotated 70S ribosomes suggests that TetM binds preferentially to the nonrotated state, i.e., analogous to the expected (posttranslocational) substrate resulting from tetracycline inhibition.”

The overall orientation of TetM structurally mimics more closely the position of EF-Tu•tRNA bound to the 70S ribosome than that of EF-G.

“The overall orientation of TetM appears rather to structurally mimic more closely the position of EF- Tu•tRNA bound to the 70S ribosome (SI Appendix, Fig. S6).”

The flipped-out conformation of A1492 and A1493 remains after the ribosome protection protein has left the ribosome.

“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.”

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Domain IV of TetM is structurally analogous to EF-G, containing a four-stranded β-sheet and two α-helices with an overall βββαβα topology.

“Sequence alignments (SI Appendix, Fig. S1) and homology modeling suggest that domain IV of TetM is structurally analogous to EF-G, containing a four-stranded β-sheet and two α-helices, with an overall βββαβα topology (Fig. 3B).”

TetM directly dislodges tetracycline to confer resistance.

“A model is presented whereby TetM directly dislodges tetracycline to confer resistance.”

• Also stated in: § Conclusion · hedged

  “In conclusion, our structure suggests that TetM confers resistance to tetracycline using a direct mechanism (Fig. 5).”

The final density map for the TetM•70S complex has a resolution of 7.2 Å, determined using the Fourier shell correlation cutoff value of 0.5.

“Further refinement of the TetM•70S complex resulted in a final density map (Fig. 1A) with a resolution of 7.2 Å (EMD-2183), as determined by using the Fourier shell correlation cutoff value of 0.5 (SI Appendix, Fig. S2B).”

The structural features imparted by TetM on the ribosome would contribute to the synergistic effect that TetM has been proposed to have on EF-Tu binding to the ribosome.

“Collectively, we believe these structural features imparted by TetM on the ribosome would contribute to the synergistic effect that TetM has been proposed to have on EF-Tu binding to the ribosome (16).”

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Mutagenesis data suggest that residues especially Y506 and Y507 within loop III of TetM are likely to be important for inducing the conformational change.

“Additionally, the mutagenesis data (Fig. 4B) suggest that residues especially Y506 and Y507 within loop III of TetM are likely to be important for inducing this conformational change.”

TetM dislodges tetracycline from its binding site on the ribosome by disrupting the reported stacking interaction between the aromatic ring D of tetracycline and the nucleobase of C1054.

“This suggests that TetM dislodges tetracycline from its binding site on the ribosome by disrupting the reported stacking interaction between the aromatic ring D of tetracycline and the nucleobase of C1054 (2), in agreement with a previous proposal (15).”

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The altered conformation of C1054, together with the flipped-out conformation of A1492 and A1493 induced by interaction with the C-terminal extension of TetM, as well as the closed conformation of the stalk base, promote rapid binding of the ternary complex EF-Tu•GTP•aa-tRNA.

“(iii) The altered conformation of C1054, together with the flipped-out conformation of A1492 and A1493 induced by interaction with the CTE of TetM, as well as the closed conformation of the stalk base, promote rapid binding of the ternary complex EF-Tu•GTP•aa-tRNA (Fig. 5C).”

TetM employs residues including Y506 and Y507 within loop III of domain IV to directly interact and alter the conformation of nucleotide C1054 within h34 of the 16S rRNA that comprises part of the tetracycline binding site.

“(i) TetM employs residues including Y506 and Y507 within loop III of domain IV to directly interact and alter the conformation of nucleotide C1054 within h34 of the 16S rRNA that comprises part of the tetracycline binding site (Fig. 5A).”

The observed binding position of tigecycline was modeled into the primary tetracycline binding site based on similarity of the chemical structures of tigecycline and tetracycline.

“Because there is no structure reported for Tgc•70S, the observed binding position of Tgc was modeled into the primary tetracycline binding site (2, 3) based on similarity of the chemical structures of the two drugs (SI Appendix, Fig. S10 D-H).”

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• AssumptionThe authors state this as an assumption: Tigecycline binds the primary tetracycline binding site in a manner similar to tetracycline, as inferred from the similarity of their chemical structures.

TetO and TetM catalyze the release of tetracycline from the ribosome in a GTP-dependent manner.

“Accordingly, TetO and TetM catalyze the release of tetracycline from the ribosome in a GTP-dependent manner (12,13).”

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GTP hydrolysis is not strictly required to dislodge tetracycline because drug release also occurs in the presence of nonhydrolyzable GTP analogues.

“Although GTPase activity is required for multiturnover of RPPs, GTP hydrolysis is however not strictly required to dislodge tetracycline because drug release also occurs in the presence of nonhydrolyzable GTP analogues (12,13).”

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The absence of secondary binding sites illustrates the increased specificity of tigecycline compared with tetracycline.

“the absence of secondary binding sites illustrates the increased specificity of Tgc compared with tetracycline.”

• AssumptionThe authors state this as an assumption: Tigecycline binds the primary tetracycline binding site in a manner similar to tetracycline, as inferred from the similarity of their chemical structures.

Crystal structures of the 30S ribosomal subunit in complex with tetracycline reveal the primary binding site to be located in helix 34 of the 16S rRNA, in a position overlapping with the anticodon stem-loop of A-site tRNA.

“Consistently, crystal structures of the small (30S) ribosomal subunit in complex with tetracycline reveal the primary binding site to be located in helix 34 (h34) of the 16S rRNA, in a position overlapping with the anticodon stem-loop of A-site tRNA (A-tRNA) (2, 3).”

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TetO was suggested to chase tetracycline from the ribosome indirectly via inducing local disturbances within h34.

“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).”

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