Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes

Abstract

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Figure 1.

Sequence alignments of HflX and HflXr proteins and MIC data. (A) Sequence alignment of the resistance-associated loop region within the N-terminal domain of selected HflX (blue) and HflXr (pink) representatives, showing independently evolved insertions in HflXr and HflX. Taxa in bold are those with both HflX and HflXr. Conserved R/Q residue (R149 in L. monocytogenes HflXr) is marked with a red asterisk. The full alignment is found in Supplementary Data S1. (B) Minimum inhibitory concentrations (MICs) of ribosome-targeting antibiotics against L. monocytogenes EGDe strains lacking or expressing HflXr or/and VgaL/Lmo0919 ARE-ABCF. The color code is made with respect to the first column that contains the ΔvgaL/pIMK3 MICs.

Figure 2.

Cryo-EM structure of the L. monocytogenes HflXr-50S complex. (A) Cryo-EM density (3 Å low-pass filtered) of the 50S ribosomal subunit (grey) with HflXr (orange). Inset shows relative orientation of (A) to crown view. (B) Isolated cryo-EM map density (grey mesh; 3 Å lowpass filtered) with a molecular model for HflXr colored according to domains: N-terminal subdomain I (NTD1, blue), N-terminal subdomain II (NTD2, red), G-domain (GD, green) and C-terminal domain (CTD, orange). (C) Interactions of HflXr (orange) NTD1 with H69 (blue), H70 (cyan), H71 (dark green), the α-helices of NTD2 with H89 (purple) and H91 (light purple), NTD2-loop with the PTC (lime) and the CTD with uL11 (light blue). (D) GDPNP (grey) in extracted density (grey mesh) within the binding pocket of the GD (orange) with a putative coordinated magnesium ion (green). (E) Binding of HflXr (orange) causes a shift in H69 (grey) compared to the vacant L. monocytogenes ribosome (light blue, aligned on 23S rRNA). (F) H69 (grey) movement caused by HflXr would sterically clash with h44 of L. monocytogenes 30S subunit (yellow, PDBID: 7NHN; (11)) on the 70S ribosome, when aligned on the basis of 23S rRNA.

Figure 3.

Interaction of the NTD subdomain II of HflXr at the PTC. (A) Arg149 of NTD2-loop of HflXr (orange) in extracted cryo-EM density. (B) HflXr loop (orange) superimposed with A- (blue) and P-site tRNAs (green)(73). (C) The loop of HflXr (orange) extends deeper into the PTC than E. coli HflX (17). Alignment in (B) and (C) are based on 23S rRNA. D-F, Binding position of HflXr NTD2-loop (orange) with Arg149 shown as sphere, superimposed with the binding site of (D) Lincomycin (Lnc, purple, PDB ID 5HKV)(57), (E) erythromycin (Ery, yellow, PDB ID 4V7U) (59) and (F) virginiamycin S1 (VgS1, blue, PDB ID 1YIT)(60). Predicted steric clashes are indicated by red lines at the overlap of spheres.

Figure 4.

Cryo-EM structure of the L. monocytogenes lincomycin-70S complex. (A) Chemical structure of lincomycin, with galactopyranosyl (orange), methylsulfanyl (yellow), hydroxypropyl (green), carboxamide (blue) and propylpyrrolidine (cyan) moieties highlighted. (B) Cryo-EM density (mesh) with molecular model of lincomycin (Lnc, blue). (C) Molecular model of Lnc (blue) with cryo-EM density (mesh) and model for waters W1-W4 (red). (D) Cryo-EM density of L. monocytogenes Lnc-70S complex with Lnc bound at the PTC, adjacent to the ribosomal NPET in the 50S (grey) subunit. (E) Lnc (blue) with surrounding waters W1-W4 (red) and 23S rRNA nucleotides (grey). (F) Water-mediated interaction of the hydroxypropyl-group of Lnc (blue) with N2 of G2094 (EcoG2061, grey) through W1 (red) and N7 of A2536 (EcoA2503, grey) through W2 (red). (G) Water-mediated interaction of Lnc (blue) with N1 of A2091 (EcoA2058, grey) and W3 and N4 of C2644 (EcoC2611, grey) with W4 (red).

Figure 5.

HflXr-induced conformational changes at the PTC are incompatible with lincomycin binding. (A) NTD2-loop Arg149 of HflXr (orange) with selected L. monocytogenes 23S rRNA nucleotides (grey). (B) as (A), but superimposed with L. monocytogenes 23S rRNA nucleotides (dark blue) from vacant 70S ribosome. Nucleotide rearrangements induced by HflXr are indicated with black arrows. (C) Comparison of L. monocytogenes 23S rRNA nucleotides in presence (green) and absence (dark blue) of lincomycin (Lnc, light blue). (D) as (A), but superimposed with Lnc (light blue) bound to the L. monocytogenes 23S rRNA nucleotides (cyan) with nucleotide rearrangements induced by HflXr indicated with black arrows and shown as spheres with red lines indicating steric clashes. (E, F) as (A), but superimposed with (E) E. coli HflX-50S complex (PDB ID 5ADY) (17), and (F) 23S rRNA nucleotides (purple) from the L. monocytogenes 70S refined into the L. monocytogenes HflX-50S complex. (G, H) Comparison of 23S rRNA nucleotides from L. monocytogenes HflX-50S complex with (G) E. coli HflX-50S complex (PDB ID 5ADY) (17), and (H) L. monocytogenes 70S ribosome.

Figure 6.

Proposed mechanism of action of HflXr. (A) An initiating 70S ribosome with initiator tRNA (teal) in the P-site (A) is stalled by an antibiotic (Lnc, red, B) with G2538 (Eco2505) and A2095 (Eco2062) in their canonical position. (C) The P-tRNA dissociates through an unknown mechanism (e.g. ArfA/ArfB, hydrolysis or tRNA drop off) which allows HflXr-GTP to recognize the resulting 70S ribosome, triggering ribosome splitting and subunit dissociation. (D) The HflXr NTD2 loop induces PTC rearrangement of G2538 (Eco2505) and A2095 (Eco2062), leading to antibiotic dissociation. (E) GTP hydrolysis allows HflXr-GDP release and the resulting free 50S subunit (F) is available for reinitiation (A).