Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding

Abstract

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/ h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.GE81112 | ribosome | X-ray crystallography | protein synthesis | antibiotics T he majority of known antibiotics target the translational apparatus (1-4), but very few (e.g., kasugamycin, edeine, pactamycin, thiostrepton) are inhibitors of the initiation phase of protein synthesis, and even fewer display specificity for either the target or the kingdom of life in which they are effective (2-8). Two notable exceptions are Furvina (9) and GE81112 (6, 10), which inhibit bacterial protein synthesis by interfering with initiator fMet-tRNA binding to the 30S ribosomal subunit, thereby preventing the formation of the 30S initiation complex (30SIC).Bacterial initiation progresses through a series of kinetic checkpoints that determine the overall efficiency and fidelity of translation initiation of a given mRNA (11-13). The initial step involves the recruitment of initiation factors (IFs) IF1, IF2·GTP, and IF3, mRNA, and fMet-tRNA to the 30S subunit that leads to the formation of the 30S preinitiation complex (30S preIC) in which initiator tRNA and mRNA are both ribosome-bound but not mutually interacting (12). Subsequently, a first-order isomerization of the 30S preIC leads to peptidyl (P)-site codon-anticodon pairing and yields a "locked" 30SIC. This 30S preIC→30SIC transition represents the first kinetic checkpoint of translational fidelity, because this process is under the kinetic control of the IFs, and during this step IF3, assisted by IF1, "inspects" the correctness of the 30S ligands, in particular the tRNA anticodon stem loop (ASL) and the mRNA start codon; any complex containing noncanonical components or having a geometry unfavorable for do...

Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin

“…Structural and kinetic analysis show that the engaged/disengaged state of the 30S subunit is also affected by the novel antibiotic GE81112, resulting in a blockade of the 30S IC progression by preventing initiation codon decoding [49]. Whether GE81112 perturbs the IF3 layout of the 30S subunit remains elusive; however, our model would suggest that the drug promotes a close distance conformation between domains.…”

Totalsynthese und Strukturkorrektur des antibiotisch wirksamen Tetrapeptids GE81112A

“…[1] Die Gruppe der GE81112-Verbindungen, die von einer Streptomyces sp.p roduziert werden, stellt eine solche Klasse potenzieller neuer Leitstrukturen dar. [2,4] Die GE81112-Antibiotika sind unter Standard-Testbedingungen gegen E. coli nahezu inaktiv und entfalten ihre volle Aktivitätnur in Minimalmedien, da sie in die Bakterien durch das Oligopeptid-Permease-System (Opp) transportiert werden. [2,3] Die GE81112-Verbindungen hemmen die Proteinsynthese durch Wechselwirkung mit der kleinen 30S-ribosomalen Untereinheit.…”

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

“…Antibacterial profiling has shown that each congener displays effective growth inhibition against several Gram‐positive and Gram‐negative pathogens . Recent studies indicated that GE81112 B stalls initiation in the unlocked 30S pre‐initiation complex state and impedes its transition to the corresponding initiation complex . This process represents a unique mechanism of action relative to other antibiotics that target the ribosome .…”