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Structural mechanism of FusB-mediated rescue from fusidic acid inhibition of protein synthesis

ABSTRACT: The antibiotic resistance protein FusB rescues protein synthesis from inhibition by fusidic acid (FA), which locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis. Here, we present time-resolved single-particle cryo-EM structures explaining the mechanism of FusB-mediated rescue. FusB binds to the FA-trapped EF-G on the ribosome, causing large-scale conformational changes of EF-G that break interactions with the ribosome, tRNA, and mRNA. This leads to dissociation of EF-G from the ribosome, followed by FA release. We also observe two independent binding sites of FusB on the classical-state ribosome, overlapping with the binding site of EF-G to each of the ribosomal subunits, yet not inhibiting tRNA delivery. The affinity of FusB to the ribosome and the concentration of FusB in S. aureus during FusB-mediated resistance support that direct binding of FusB to ribosomes could occur in the cell. Our results reveal an intricate resistance mechanism involving specific interactions of FusB with both EF-G and the ribosome, and a non-canonical release pathway of EF-G.

INSTRUMENT(S):

ORGANISM(S): Staphylococcus Aureus

TISSUE(S): Cell Culture

SUBMITTER: Johannes Fuchs

LAB HEAD: Carina Sihlbom Wallem

PROVIDER: PXD062129 | Pride | 2025-05-07

REPOSITORIES: Pride

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			Action	DRS
	5046_TMT_Eclipse_241001_31.msf	Msf
	5046_TMT_Eclipse_241001_31_FIN.msf	Msf
	5046_TMT_Lumos_241004_14_15_FIN.msf	Msf
	5046_TMT_Lumos_241004_18_19_FIN.msf	Msf
	5046_TMT_Lumos_241004_22_23_FIN.msf	Msf

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Structural mechanism of FusB-mediated rescue from fusidic acid inhibition of protein synthesis.

González-López Adrián A Ge Xueliang X Larsson Daniel S D DSD Sihlbom Wallem Carina C Sanyal Suparna S Selmer Maria M

Nature communications 20250418 1

The antibiotic resistance protein FusB rescues protein synthesis from inhibition by fusidic acid (FA), which locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis. Here, we present time-resolved single-particle cryo-EM structures explaining the mechanism of FusB-mediated rescue. FusB binds to the FA-trapped EF-G on the ribosome, causing large-scale conformational changes of EF-G that break interactions with the ribosome, tRNA, and mRNA. This leads to dissociation of EF-G from the ...[more]

PMID: 40251147

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Project description:Methicillin resistant Staphylococcus aureus (MRSA) infection is becoming refractory to existing antibiotic therapy owing to the inherent ability of S. aureus to develop rapid resistance and is considered a major threat to public health. We found that a natural isolate of Bacillus pumilus from the Columbia River Estuary produces a strong anti-MRSA compound, amicoumacin A. As amicoumacin A has been reported to exhibit anti-microbial, anti-inflammatory, and anti-ulcer activities, we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in the expression of genes involved in several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which has been shown to be induced in response to a collapse of membrane potential. In order to gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by serial passage method was carried out. Single point mutations resulting in codon substitutions were uncovered in several distinct genes: ksgA, RNA dimethyltranferase; fusA, elongation factor G; dnaG, primase, ; lacD, tagatose 1,6-bisphosphate aldolase, ; and SACOL0611, encoding a putative glycosyl transferase gene. Based on these results, a candidate approach was undertaken to recreate the same amino acid substitution individually in FusA and KsgA, each of which resulted in two-fold resistance towards amicoumacin A. The fusA gene is known as the site for fusidic acid- resistant mutations; however the codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance occur in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability. Amicoumacin A, isolated from Bacillus pumilus, was added to exponentially growing cultures (OD600 =0.5) of Staphylococcus aureus COL at concentrations leading to around 12% and 20% reduction of OD600 after 10 min and 40 min, respectively. Total RNA was isolated from three biological replicates. Labeled cDNA from treated and control cultures (Cy5) was hybridized against a common reference cDNA pool (Cy3). The reference pool was prepared from a mixture of equal amounts of total RNA isolated from all stress and control samples in the experiment.