Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin

Abstract

Background: The bacterial ribosome is a primary target of several classes of antibiotics. Investigation of the structure of the ribosomal subunits in complex with different antibiotics can reveal the mode of inhibition of ribosomal protein synthesis. Analysis of the interactions between antibiotics and the ribosome permits investigation of the specific effect of modifications leading to antimicrobial resistances. Streptogramins are unique among the ribosome-targeting antibiotics because they consist of two components, streptogramins A and B, which act synergistically. Each compound alone exhibits a weak bacteriostatic activity, whereas the combination can act bactericidal. The streptogramins A display a prolonged activity that even persists after removal of the drug. However, the mode of activity of the streptogramins has not yet been fully elucidated, despite a plethora of biochemical and structural data.

Results: The investigation of the crystal structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with the clinically relevant streptogramins quinupristin and dalfopristin reveals their unique inhibitory mechanism. Quinupristin, a streptogramin B compound, binds in the ribosomal exit tunnel in a similar manner and position as the macrolides, suggesting a similar inhibitory mechanism, namely blockage of the ribosomal tunnel. Dalfopristin, the corresponding streptogramin A compound, binds close to quinupristin directly within the peptidyl transferase centre affecting both A- and P-site occupation by tRNA molecules.

Conclusions: The crystal structure indicates that the synergistic effect derives from direct interaction between both compounds and shared contacts with a single nucleotide, A2062. Upon binding of the streptogramins, the peptidyl transferase centre undergoes a significant conformational transition, which leads to a stable, non-productive orientation of the universally conserved U2585. Mutations of this rRNA base are known to yield dominant lethal phenotypes. It seems, therefore, plausible to conclude that the conformational change within the peptidyl transferase centre is mainly responsible for the bactericidal activity of the streptogramins and the post-antibiotic inhibition of protein synthesis.

Figure 1

Interactions of streptogramins with 23S rRNA. (A) Chemical structure of quinupristin and dalfopristin. The hydrogen bonds towards 23S rRNA nucleotides are indicated. (B) Overview of the nucleotides involved in binding in comparison with those indicated by various biochemical and genetic experiments [5, 7, 12-30]. Both images contain numbering for E. coli (in green) and for D. radiodurans (in red). The sequence itself corresponds to 23S rRNA of D. radiodurans. All other images use numbering according to E. coli.

Figure 2

Structure of dalfopristin and quinupristin within the PTC. To facilitate visualization of the interactions of dalfopristin and quinupristin with 23S rRNA, rRNA bases not involved in binding have been omitted. (A) Local environment of dalfopristin (in orange). A2062 is highlighted in purple; nucleotides, which are interacting through hydrogen bonds with either dalfopristin or quinupristin, are shown in dark blue. (B) Local environment of quinupristin (in green). Colours are as in (A). (C) Stereo view of the electron density map of quinupristin (in green) and dalfopristin (in orange). Both compounds have been omitted during calculation of the sigmaA weighted difference map, which is contoured at 1.5σ. (D) Stereo representation of dalfopristin and quinupristin and their local environment. Colours as in (A).

Figure 3

Comparison of antibiotic binding sites. (A) To visualize the relative orientations of different classes of antibiotics and substrates compared with dalfopristin and quinupristin, several structures have been aligned and overlaid: clindamycin (PDB entry 1JZX), erythromycin (1JZY), chloramphenicol (1K01) and CC-Puromycin molecules in A- (A-CCPuro) and P-site (P-CCPuro). CC-Puromycin coordinates were taken from Bashan et al. (2003) [40]. (B) Overview of the binding sites of quinupristin and dalfopristin within the 50S ribosomal subunit, in relation to the P-site tRNA and the ribosomal exit tunnel (highlighted in gold).

Figure 4

Conformation of the PTC. (A) Different orientations of A2062 and its local environment for native D50S (purple), for the complex with Synercid^® (blue) and for the H50S streptogramin complex (yellow). (B) Comparison of the position of dalfopristin in D50S (orange) with the position of virginiamycin M in H50S (light green), and the corresponding folds of 23S rRNA in the vicinity of U2585 (in blue for D50S and in yellow for H50S). (C) Structure and electron density around U2585. For comparison, the orientation of U2585 in the native D50S structure is also shown. The electron density is a sigmaA weighted difference map omitting the whole peptidyl-transferase ring from the calculation. (D) Local structure around U2585 overlaid with the native structure; putative hydrogen bonds of U2585 in its new orientation are indicated. (E) Approximate energy profile derived from modelling intermediate conformations, units being arbitrary. N indicates the energy of the native conformation and S the one of the complex with Synercid^®.