Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Structural and Biochemical Studies of Antibiotic Resistance and Ribosomal Frameshifting
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, 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 [en]
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: urn:nbn:se:uu:diva-205131ISBN: 978-91-554-8728-7 (print)OAI: oai:DiVA.org:uu-205131DiVA: diva2:641588
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
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. Crystal structure of AadA at 2.5 Å resolution - an aminoglycoside 3" adenyltransferase
Open this publication in new window or tab >>Crystal structure of AadA at 2.5 Å resolution - an aminoglycoside 3" adenyltransferase
(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-205128 (URN)
Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2016-11-30
4. tRNASer and tRNAThr induce -1 frameshifting using alternative anticodon-loop structures
Open this publication in new window or tab >>tRNASer and tRNAThr induce -1 frameshifting using alternative anticodon-loop structures
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biology
Identifiers
urn:nbn:se:uu:diva-205130 (URN)
Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2014-01-22

Open Access in DiVA

fulltext(12026 kB)1979 downloads
File information
File name FULLTEXT01.pdfFile size 12026 kBChecksum SHA-512
8e6b09ca0ff98398468e4b3f9b6d3536cb043eb827c494cf3ffb41c7b517e65fad9a3d4fefbd13312707bf4b8fd5e6648b05cd6bbc5d5a1cfc8f0b4260511b32
Type fulltextMimetype application/pdf