Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Xiaohu Guo,Maria Selmer,Suparna Sanyal,Ravi Kiran Koripella,Kristina Bäckbro,Yang Chen,Kristin Peisker,Chandra Sekhar Mandava

doi:10.1098/rsob.120016

Abstract

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.

Highlights

Fusidic acid (FA) is a bacteriostatic antibiotic that was first isolated from the fungus Fusidium coccineum in the early 1960s [1]
An N-terminally His-tagged version of FusB was cloned from plasmid pUB101 present in clinical isolates of S. aureus [21] and overexpressed in E. coli
FusB was crystallized under several different conditions at the high-throughput crystallization facility in Grenoble, France

Summary

Introduction

Fusidic acid (FA) is a bacteriostatic antibiotic that was first isolated from the fungus Fusidium coccineum in the early 1960s [1]. FA blocks bacterial protein synthesis by locking elongation factor G (EF-G) to the ribosome [2]. FA is mainly used against staphylococcal infections, often in combination with other drugs to prevent resistance development. EF-G is a translational GTPase catalysing two different steps of protein synthesis (reviewed by Schmeing & Ramakrishnan [3]). EF-G is needed for translocation of tRNAs and mRNA with respect to the ribosomal 30S subunit to make a new mRNA codon available for decoding. EF-G acts together with ribosome recycling factor (RRF) in splitting of the ribosomal posttermination complex. In both of these steps, GTP hydrolysis by EF-G is used as an energy source, and in both cases FA prevents the release of EF-G from

Methods

Results

Discussion

Conclusion