Interaction of the CMLAL leader peptide with E. coli ribosome, binding chloramphenicol in non-canonical site

With the use of docking, equilibrium and biased molecular dynamics simulations, the structure of the 70S E. coli A/A,P/P-ribosome complex with chloramphenicol, an antibiotic, bound in a non-canonical site near the peptidyl transferase center, and ClmAL leader peptide, placed in the nascent peptide exit tunnel, has been obtained. In this structure, chloramphenicol is retained in the ribosome due to wedging of its nitrophenyl residue into the cavity between the Ψ2504 and U2506 residues of 23S rRNA and formation of hydrophobic contacts with them, as well as hydrogen bonds with the G2505 and G2061 residues of 23S rRNA. The ClmAL leader peptide forms many stable hydrogen bonds with the G2061, m2A2503, U2609, and C2610 residues of 23S rRNA. Molecular dynamics simulations of this ternary complex has shown that the mechanism of antibiotic action is to induce divergence of the peptidyl transferase reaction of the substrates relative to each other at a distance that excludes the transpeptidation reaction. This divergence of the peptidyl transferase reaction substrates is stabilized by interactions between the side chain amino group of the lysine residue in the A site, on the one hand, and the C2063 base and chloramphenicol, on the other hand. In this case, the α-amino group of the lysine residue forms a hydrogen bond with the carbonyl group of the Ala-7 CmlAL residue. Together with specific interactions of the CmlAL peptide residues with the rRNA residues of the nascent peptide exit tunnel, this explains the arrest of translation in the presence of chloramphenicol at this particular sequence and, as a consequence, the presence of this sequence in the genes encoding the CmlA transporter protein, which is responsible for ribosomal resistance to this antibiotic.