Conjugates of Amino Acids and Peptides with 5-O-Mycaminosyltylonolide and Their Interaction with the Ribosomal Exit Tunnel

Abstract

During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

“…Binding affinities of CAM-derivatives to E. coli ribosomes was analyzed by competition binding assay using fluorescently-labeled BODIPY-ERY as described before [26, 27, 37]. BODIPY-ERY (4 nM) was incubated with ribosomes (25 nM) for 30 min at 25°C in the buffer containing 20 mM HEPES-KOH (pH 7.5), 50 mM NH 4 Cl, 10 mM Mg(CH 3 COO) 2 , and 0.05% Tween-20.…”

Molecular dynamics investigation of a mechanism of allosteric signal transmission in ribosomes

“…Controlled dynamics and meta dynamics were calculated using the PLUMED software [45] version 1.3 that works together with the GROMACS software suite. Position and frequency of hydrogen bonds were analyzed as described earlier [33].…”

“…In particular, it revealed some potential contacts of the key amino acid residues within peptides that arrest translation with senso ry nucleotide residues of the RT [32]. Recently, using this method, we found possible reasons for abnormal biologi cal activity of peptide derivatives of macrolides, which inhibit protein synthesis, and described a novel potential site in the walls of the RT that interacts with nascent polypeptide chains [33]. By using MD simulation, we observed that the potential signal transmitting pathway A2058 C2063 behaved as a dynamic ensemble of interde pendent conformational states, wherein cascade like changes can originate.…”

Macrolide-Peptide Conjugates as Probes of the Path of Travel of the Nascent Peptides through the Ribosome

“…19,20 Specifically, molecular dynamics simulations suggest that the N-acylglycyl moiety of the aminoacylated 5-O-mycominosyl-tylonolide derivative could be positioned to form hydrogen bonds with 23S rRNA A752 and Lys 90 of ribosomal protein L22. These hydrogen bonds could potentially disrupt the essential A752:U2609 base pair, which has previously been shown to play a role in both peptide stalling and macrolide antibiotic interactions.…”