title: Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin.
abstract: Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR)Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.
authors.affiliation: Centro de Investigación e Innovación, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas-UPC, Lima L-33, Peru. robertochulluncuy1@gmail.com.
authors.forename: Roberto
authors.initials: R
authors.lastname: Chulluncuy
authors.affiliation: Centro de Investigación e Innovación, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas-UPC, Lima L-33, Peru. carlosespiche852@gmail.com.
authors.forename: Carlos
authors.initials: C
authors.lastname: Espiche
authors.affiliation: Centro de Investigación e Innovación, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas-UPC, Lima L-33, Peru. jose.nakamoto@upch.pe.|Facultad de Ciencias y Filosofía Alberto Cazorla Talleri, Universidad Peruana Cayetano Heredia-UPCH, Lima L-31, Peru. jose.nakamoto@upch.pe.
authors.forename: Jose Alberto
authors.initials: JA
authors.lastname: Nakamoto
authors.affiliation: Laboratory of Genetics, Department of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy. attilio.fabbretti@unicam.it.
authors.forename: Attilio
authors.initials: A
authors.lastname: Fabbretti
authors.affiliation: Centro de Investigación e Innovación, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas-UPC, Lima L-33, Peru. pmilon@upc.pe.
authors.forename: Pohl
authors.initials: P
authors.lastname: Milón
keywords: 30S subunit; FRET; IF3; kanamycin; streptomycin; translation initiation; tuberculosis