ErmBL Translation on the Ribosome in the Presence of Erythromycin is Stalled by Inhibition of Peptide Bond Formation

Abstract

Translation of the Streptococcus sanguis ErmBL peptide is stalled in the presence of the antibiotic erythromycin, which binds inside the ribosomal exit tunnel. The stalling regulates the expression of the downstream gene of the resistance methyltransferase ErmB. Here, we investigate the stalling mechanism by explicit-solvent all-atom molecular dynamics simulations of the ribsome-ErmBL complex in the presence and absence of erythromycin. The simulations, totaling 12 µs simulation time, were started from a 3.6-A-resolution cryo-EM structure. In the cryo-EM structure the ribose of A76 of the peptidyl tRNA is shifted from its canonical pre-attack position. In the simulations without erythromycin, the ErmBL peptide explores conformations, which would overlap with the antibiotic. Together, these two findings show that erythromycin actively restrains the conformation of the peptide inside the tunnel and leads to the shift of the A76 ribose. Further, 23S nucleotides 2504-2506 and 2452 adopt different conformations depending on the presence of erythromycin. These nucleotides form a part of the A site cavity and are shifted away from the P site in the presence of erythromycin. In the stalled complex, the A-site tRNA carries a charged lysine which in the simulations, interacts with these nucleotides and follows their movement. This movement in turn, increases the distance between the attacking alpha-amino group of the lysine and the carbonyl-carbon of the peptide by more than 1 A, suggesting a lower rate of peptide bond formation. Upon mutation of the lysine to an alanine, we observe a decrease of this distance. This decrease is in agreement with the observation that the mutation alleviates stalling. On the basis of the results, we propose that a mutation of the lysine to an arginine, would be expected to preserve stalling.