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Life will find a way: Structural and evolutionary insights into FusB and HisA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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 [en]
HisA, TrpF, protein evolution, bi-functional enzyme, fusidic acid, antibiotic resistance, protein synthesis, FusB
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-265718ISBN: 978-91-554-9409-4 (print)OAI: oai:DiVA.org:uu-265718DiVA: diva2:866444
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
List of papers
1. Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
Open this publication in new window or tab >>Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 41, 24657-24668 p.Article in journal (Refereed) Published
Abstract [en]

HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-260701 (URN)10.1074/jbc.M115.678086 (DOI)000362598300003 ()26294764 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 283570
Available from: 2015-08-23 Created: 2015-08-23 Last updated: 2017-12-04
2. 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
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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.

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
3. Characterization of interactions in FusB-mediated fusidic acid resistance
Open this publication in new window or tab >>Characterization of interactions in FusB-mediated fusidic acid resistance
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-265549 (URN)
Available from: 2015-11-01 Created: 2015-11-01 Last updated: 2016-01-13
4. Functional and structural innovations in the real-time evolution of new genes
Open this publication in new window or tab >>Functional and structural innovations in the real-time evolution of new genes
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-265716 (URN)
Available from: 2015-11-02 Created: 2015-11-02 Last updated: 2016-01-13

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