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Characterizing Elongation of Protein Synthesis and Fusidic Acid Resistance in Bacteria
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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 [en]
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: urn:nbn:se:uu:diva-207924ISBN: 978-91-554-8761-4 (print)OAI: oai:DiVA.org:uu-207924DiVA: diva2:650357
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
List of papers
1. Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
Open this publication in new window or tab >>Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 18, 3789-3803 p.Article in journal (Refereed) Published
Abstract [en]

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 A resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the individual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 A relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46-56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694-699]. The mutations can be classified as affecting FA binding, EF-G-ribosome interactions, EF-G conformation, and EF-G stability.

Keyword
antibiotic resistance, crystallography, elongation factor G (EF-G), fusidic acid, switch region
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-134901 (URN)10.1111/j.1742-4658.2010.07780.x (DOI)000281555600016 ()
Available from: 2010-12-02 Created: 2010-12-02 Last updated: 2017-12-12Bibliographically approved
2. Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus
Open this publication in new window or tab >>Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus
Show others...
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 36, 30257-30267 p.Article in journal (Refereed) Published
Abstract [en]

Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify 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 a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.

Keyword
Antibiotic Resistance, GTPase, Staphylococcus aureus, Translation, Translation Elongation Factors
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-182775 (URN)10.1074/jbc.M112.378521 (DOI)000308579800019 ()
Available from: 2012-10-17 Created: 2012-10-15 Last updated: 2017-12-07Bibliographically approved
3. Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
Open this publication in new window or tab >>Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
Show others...
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.

Keyword
FusB, elongation factor G, fusidic acid, antibiotic resistance
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-181147 (URN)10.1098/rsob.120016 (DOI)000307111800002 ()
Available from: 2012-09-18 Created: 2012-09-17 Last updated: 2016-01-13Bibliographically approved
4. Essential role of Histidine 92 in elongation factor-G in GTP hydrolysis and inorganic phosphate release during elongation of protein synthesis
Open this publication in new window or tab >>Essential role of Histidine 92 in elongation factor-G in GTP hydrolysis and inorganic phosphate release during elongation of protein synthesis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The histidine (H) residue at the apex of switch II is conserved in all translational GTPases. Thishistidine (H92) in elongation factor G (EF-G) has been implicated in GTP hydrolysis andinorganic phosphate (pi) release similar to H85 in elongation factor-Tu (EF-Tu). Mutagenesis ofH92 to alanine (A) and glutamic acid (E) showed different degrees of defect in different steps ofelongation. While H92A was ~7 times slower than wild type EF-G in ribosome mediated GTPhydrolysis, it was 100 times slower in both pi release and tRNA translocation. The H92E mutant,on the other hand, was 100 times slower in all these steps. Both mutants were significantlydefective (~1000 times slower) in tripeptide formation that which requires dissociation of EF-Gfrom the post-translocation state. Thus, our results indicate that GTP hydrolysis takes place priorto tRNA translocation, whereas Pi release occurs probably after or independent of thetranslocation step. Since translocation involves back ratcheting of the ribosomal subunits ourresults suggest that there is a cross-talk between GTP hydrolysis by EF-G and ribosomal subunitrotation. We further confirm that Pi release is essential for the next round of elongation.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-207882 (URN)
Available from: 2013-09-20 Created: 2013-09-20 Last updated: 2017-01-25

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Koripella, Srihari Nagendra Ravi Kiran

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