Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity

Abstract

New insights into functional flexibility at the peptidyl transferase center (PTC) and its vicinity were obtained by analysis of pleuromutilins binding modes to the ribosome. The crystal structures of Deinococcus radiodurans large ribosomal subunit complexed with each of three pleuromutilin derivatives: retapamulin (SB-275833), SB-280080, and SB-571519, show that all bind to the PTC with their core oriented similarly at the A-site and their C14 extensions pointing toward the P-site. Except for an H-bond network with a single nucleotide, G2061, which involves the essential keto group of all three compounds, only minor hydrophobic contacts are formed between the pleuromutilin C14 extensions and any ribosomal component, consistent with the PTC tolerance to amino acid diversity. Efficient drug binding mode is attained by a mechanism based on induced-fit motions exploiting the ribosomal intrinsic functional flexibility and resulting in conformational rearrangements that seal the pleuromutilin-binding pocket and tightens it up. Comparative studies identified a network of remote interactions around the PTC, indicating that pleuromutilins selectivity is acquired by nonconserved nucleotides residing in the PTC vicinity, in a fashion resembling allosterism. Likewise, pleuromutilin resistant mechanisms involve nucleotides residing in the environs of the binding pocket, consistent with their slow resistance-development rates.

Fig. 1.

Chemical formula of selected pleuromutilin derivatives. Typically, the tricyclic mutilin core is conserved among pleuromutilin derivatives. Notable is the variability of the C14 extension (R₁ in the large box). Large box, previously studied pleuromutilins (Left); pleuromutilins characterized in the current study (Right); mutilin and the natural pleuromutilin (Upper Left); acyl-carbamate semisynthetic derivatives (Lower). Notable is the conserved C21 carbonyl that is crucial for pleuromutilins activity (3, 16). (Upper Right) Small box, R₂, the acyl-carbamate derivative SB-571519 includes an additional hydroxyl that substituted on C2.

Fig. 2.

Selected regions from the crystal structures of the large ribosomal subunit from D. radiodurans complexed with pleuromutilin acyl-carbamate derivative SB-571519 (in red: a, d, and g) and two sulfanyl-acetate derivatives retapamulin (SB-275833) (in yellow: b, e, and h) and SB-280080 (in pink: c, f, and i) at 3.50, 3.66, and 3.56-Å resolution, respectively. (a–c) 2 F_o − F_c electron density maps, contoured at 1.5 σ level. (d–f) Interactions between pleuromutilin derivatives and 23S rRNA (the 23S rRNA bound conformation is shown in dark blue for SB-571519, orange for SB-275833, and cyan for SB-28008). All show H-bonds between G2061 and pleuromutilins' C14 extension and the possibility of H-bond between C11 hydroxyl and G2505 phosphate. In SB-571519 and SB-280080 complexes, C11 hydroxyl may serve as acceptor in an additional H-bond with the OH group of A2503 O2′, and a nontypical H-Bond is observed between U2585 and U2506. (g–i) Induced-fit mechanism promotes pleuromutilins binding (color code as in d–f). In all, the unbound 23S rRNA (PDB ID code 1NKW) is black, and the pleuromutilin-bound conformations are colored. On binding, U2585 translates away from the C14 extension and U2506 rotates toward the pleuromutilin and thus closes tightly the binding pocket. H-bonds (d and f) or other interactions (e) between the two shifted nucleotides may further stabilize the rRNA conformation at the bound state.

Fig. 3.

Four pleuromutilin derivatives superimposed in the binding pocket. (a) A surface representation of the binding pocket. Several nucleotides have been removed to permit a clear view of the binding site. The structure of D50S/SB-517519 was used for surface representation. (b) A side view of pleuromutilins with the 3′ ends of an A-site tRNA mimic and the derived P-site tRNA (31). All pleuromutilin derivatives presented here are located at the PTC, with their tricyclic core oriented similar to tiamulin (22) and their C14 extensions placed within the PTC.

Fig. 4.

Remote factors acquiring resistance and selectivity. (a–c) Possible contribution of L3 to pleuromutilin resistance. (a) 2 F_o – F_c electron density map of the carbamate-derivative (red) SB-571519. 23S rRNA nucleotides are shown in black, and protein L3 Arg144 in green. Blue mesh is contoured at 1.0σ. The red meshes (contoured at 5.0σ) indicate phosphate locations. (b) Multiple sequence alignment of protein L3 from selected bacteria. Top numbering according to E. coli (where Arg144 of D. radiodurans is Asn149), bottom bars indicate conservation among these strains, and yellow circles indicate mutations with reduced pleuromutilin susceptibility in E. coli (30), S. pyogenes, S. aureus (12), B. pilosicoli, and B. hyodysenteriae (31). Most of these mutations are located in a highly diverse 6-aa window (orange box). In D. radiodurans, this window includes Arg144 (green square). (c) L3 protein penetrate from the surface of the ribosome deeply toward the vicinity of the PTC (right image, L3 in green, SB-571519 in red). The L3 nonconserve loop-like region (consisting of six amino acids, shown in b in the orange box) is colored yellow in the center and left image. In D. radiodurans, Arg144 electrostatically interacts with U2506 phosphate (a). 23S rRNA nucleotides 2504 to 2506 (left, gray surface representation) define a large portion of induced-fit binding pocket, obtained mainly by conformational change of U2506 (see Fig. 2 g–i and Induced-Fit Mechanism for Pleuromutilin Binding). (d) Differences in remote nucleotides acquire pleuromutilin selectivity. An example is U2504, which is among the nucleotides that define pleuromutilins binding site. In eubacteria it points toward the PTC, whereas in the Archaeon H50S, this nucleotide stacks with 2055, which is A in eukarya and archaea but C in eubacteria. This stacking seems to stabilize U2504 position away from the binding pocket. SB-571519 is red; nucleotides 2504 and 2055 are blue in its complex with D50S and purple in H50S.