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Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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.

Place, publisher, year, edition, pages
2010. Vol. 277, no 18, 3789-3803 p.
Keyword [en]
antibiotic resistance, crystallography, elongation factor G (EF-G), fusidic acid, switch region
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-134901DOI: 10.1111/j.1742-4658.2010.07780.xISI: 000281555600016OAI: oai:DiVA.org:uu-134901DiVA: diva2:374051
Available from: 2010-12-02 Created: 2010-12-02 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Structural and Biochemical Studies of Antibiotic Resistance and Ribosomal Frameshifting
Open this publication in new window or tab >>Structural and Biochemical Studies of Antibiotic Resistance and Ribosomal Frameshifting
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation.

Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome.

Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer.

Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen.

These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 66 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1064
Keyword
protein synthesis, elongation, elongation factor G, fusidic acid, antibiotic resistance, aminoglycoside adenyltransferase, ribosomal frameshifting
National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-205131 (URN)978-91-554-8728-7 (ISBN)
Public defence
2013-10-04, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2013-09-12 Created: 2013-08-14 Last updated: 2014-01-22
2. 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

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