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Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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2012 (English)In: Open Biology, ISSN 2046-2441, Vol. 2, 120016- p.Article in journal (Refereed) Published
Abstract [en]

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 angstrom 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.

Place, publisher, year, edition, pages
2012. Vol. 2, 120016- p.
Keyword [en]
FusB, elongation factor G, fusidic acid, antibiotic resistance
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-181147DOI: 10.1098/rsob.120016ISI: 000307111800002OAI: oai:DiVA.org:uu-181147DiVA: diva2:553125
Available from: 2012-09-18 Created: 2012-09-17 Last updated: 2016-01-13Bibliographically approved
In thesis
1. Characterizing Elongation of Protein Synthesis and Fusidic Acid Resistance in Bacteria
Open this publication in new window or tab >>Characterizing Elongation of Protein Synthesis and Fusidic Acid Resistance in Bacteria
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1079
Keyword
Ribosome, Elongation factor-G, FusB, GTP, Staphylococcus aureus, Escherichia coli and Fusidic acid
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-207924 (URN)978-91-554-8761-4 (ISBN)
Public defence
2013-10-25, B21, BMC, Husargatan, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-10-04 Created: 2013-09-20 Last updated: 2014-01-23
2. Life will find a way: Structural and evolutionary insights into FusB and HisA
Open this publication in new window or tab >>Life will find a way: Structural and evolutionary insights into FusB and HisA
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

How do microbes adapt to challenges from the environment? In this thesis, two distinct cases were examined through structural and biochemical methods. In the first, we followed a real-time protein evolution of HisA to a novel function. The second case was fusidic acid (FA) resistance mediated by the protein FusB in Staphylococcus aureus.

In the first study, the aim was to understand how mutants of HisA from the histidine biosynthetic pathway could evolve a novel TrpF activity and further evolve to generalist or specialist enzymes. We solved the crystal structure of wild type Salmonella enterica HisA in its apo-state and the structures of the mutants D7N and D7N/D176A in complex with the substrate ProFAR. These two distinct complex structures showed us the coupled conformational changes of HisA and ProFAR before catalysis. We also solved crystal structures of ten mutants, some in complex with substrate or product. The structures indicate that bi-functional mutants adopt distinct loop conformations linked to the two functions and that mutations in specialist enzymes favor one of the conformations. We also observed biphasic relationships in which small changes in the activities of low-performance enzymes had large effects on fitness, until a threshold, above which large changes in enzyme performance had little effect on fitness.

Fusidic acid blocks protein translation by locking elongation factor G (EF-G) to the ribosome after GTP hydrolysis in elongation and recycling of bacterial protein synthesis. To understand the rescue mechanism, we solved the crystal structure of FusB at 1.6Å resolution. The structure showed that FusB is a two-domain protein and C-terminal domain contains a treble clef zinc finger. Using hybrid constructs between S. aureus EF-G that binds to FusB, and E. coli EF-G that does not, the binding determinants were located to domain IV of EF-G. This was further supported by small-angle X-ray scattering studies of the FusB·EF-G complex. Using single-molecule methods, we observed FusB frequently binding to the ribosome and rescue of FA-inhibited elongation by effects on the non-rotated state ribosome. Ribosome binding of FusB was confirmed by isothermal titration calorimetry.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 68 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1317
Keyword
HisA, TrpF, protein evolution, bi-functional enzyme, fusidic acid, antibiotic resistance, protein synthesis, FusB
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-265718 (URN)978-91-554-9409-4 (ISBN)
Public defence
2015-12-18, B41, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2015-11-25 Created: 2015-11-02 Last updated: 2016-01-13

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Guo, XiaohuPeisker, KristinBäckbro, KristinaChen, YangKoripella, Ravi KiranMandava, Chandra SekharSanyal, SuparnaSelmer, Maria

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