Structural basis for cross-resistance to ribosomal PTC antibiotics

Abstract

Clinically relevant antibiotics that target the ribosomal peptidyl transferase center (PTC), a highly conserved ribosomal region, exert their inhibitory action by exploiting the flexibility of PTC nucleotides, which trigger modulations of the shape of the antibiotic binding pocket. Resistance to these antibiotics was observed clinically and in vitro. Based on the crystal structures of the large ribosomal subunit from eubacterium suitable to represent pathogens in complex with these antibiotics, it was found that all nucleotides mediating resistance to PTC antibiotics cluster on one side of the PTC. Over half of the nucleotides affecting resistance reside in regions of lower sequence conservation, and are too distal to make Van der Waals interactions with the bound drugs. Alterations of the identity of these nucleotides may not lethally affect ribosome function, but can hamper antibiotic binding through changes in the conformation and flexibility of specific PTC nucleotides. Comparative analysis revealed properties likely to lead to cross-resistance and enabled their parameterization. As the same nucleotides are frequently involved in resistance to more than a single family of antibiotics, the common pattern explains medically observed cross-resistance to PTC antibiotics and suggests the potential for a wider clinical threat.

Fig. 1.

The antibiotics binding pockets within the PTC. (A) 2D diagram of the 23S RNA at the vicinity of the PTC. Green arrows indicate nucleotides mediating resistance to PTC antibiotics. Arrow size is proportional to the number of different classes of antibiotics that are being affected. Relations between E. coli cell vitality and nucleotide alterations (30) are color coded. Absolutely essential nucleotide, which cannot be mutated are shown in red. Nucleotides that can be replaced by a single nucleotide are shown in orange. Nucleotides that can be replaced by 2 or 3 other nucleotides are shown in yellow. Nomenclature used as established (31). Specific sections of the 2D diagram have colors identical to the colors of their corresponding regions in the 3D structure shown in B. (B) The 3-dimensional positions of PTC antibiotics showing their overlapping positions. The antibiotics chloramphenicol, clindamycin, retapamulin, dalfopristin, and linezolid are shown in yellow, cyan, orange, magenta, and pink, respectively. A site tRNA 3′ end and the derived P site tRNA (32, 33) are shown in transparent blue and green, respectively. The 23S rRNA is shown in red, yellow, and blue, as their corresponding sections in the 2D representation (A). 23S rRNA segments not shown in A are colored gray. The black arc in A is not shown in B because the latter is shown from the front wall direction.

Fig. 2.

Nucleotides shown to undergo mutations or methylations that confirm PTC antibiotic resistance or reduce susceptibility. Nucleotides are colored according to the number of affected classes (1, 2, 3, 4, and 5 classes are represented by gray, yellow, orange, red, and black, respectively). The pink surface shows the total volume occupied by the PTC antibiotics: clindamycin (lincosamides), dalfopristin (streptogramins_A), retapamulin (pleuromutilins), chloramphenicol (phenicols), and linezolid (oxazolidinones). For orientation, the A site tRNA 3′ end and the derived P site tRNA 3′ end (32, 33) are shown in blue and green, respectively. Images were taken from the direction of the L7/12 stalk (A and B) and from the top of the cavity leading to the PTC (C and D). Nucleotides located in the vicinity of the PTC within a distance <8 Å from the corresponding antibiotic, and are not involved in known resistance determinants, are either shown as wheat-colored lines (B and D) or excluded (A and C).

Fig. 3.

Overlaps of resistance determinants and distances between antibiotics binding sites and nucleotides mediating resistance. (A) Boxplot representation of nucleotide-antibiotic distances determined for D50S and H50S complexes. Dotted lines show medians; the upper and lower horizontal lines of the boxes stand for upper and lower quartiles (namely cutoffs for 25% and 75% of the data). Top and bottom external horizontal lines show the maximal and minimal values. (B) Resistant mutations or methylations observed in bacterial strains including (black bars) or excluding (gray bars) archaea (–36). x axis indicates the number of different classes of antibiotics that are being affected. y axis stands for the number of nucleotides characterized for this observation.

Fig. 4.

Remote mutations that affect the conformation and/or the flexibility of U2504 by a network of interactions. (A) PTC antibiotics chloramphenicol (yellow), clindamycin (cyan), retapamulin (orange), dalfopristin (magenta) and linezolid (pink) bind in close proximity to U2504 (red). (B–G) Shown are selected interactions within the networks around U2504. rRNA is gray, pink, or orange for D50S, H50S, or T70S, respectively. Wherever drawn, U2504 of D50S is red. Images of D50S, T70S, and H50S were generated from their coordinates (PDB ID codes 1NKW, 2J01, and 1S72, respectively).