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dc.contributor.author
Roderer, Kathrin
dc.contributor.author
Neuenschwander, Martin
dc.contributor.author
Codoni, Giosiana
dc.contributor.author
Sasso, Severin
dc.contributor.author
Gamper, Marianne
dc.contributor.author
Kast, Peter
dc.date.accessioned
2018-11-01T10:32:33Z
dc.date.available
2017-06-11T15:35:12Z
dc.date.available
2018-11-01T10:32:33Z
dc.date.issued
2014-12-31
dc.identifier.issn
1932-6203
dc.identifier.other
10.1371/journal.pone.0116234
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/96603
dc.identifier.doi
10.3929/ethz-b-000096603
dc.description.abstract
The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
Public Library of Science
en_US
dc.rights.uri
http://creativecommons.org/licenses/by/4.0/
dc.title
Functional mapping of protein-protein interactions in an enzyme complex by directed evolution
en_US
dc.type
Journal Article
dc.rights.license
Creative Commons Attribution 4.0 International
ethz.journal.title
PLoS ONE
ethz.journal.volume
9
en_US
ethz.journal.issue
12
en_US
ethz.journal.abbreviated
PLoS ONE
ethz.pages.start
e116234
en_US
ethz.size
27 p.
en_US
ethz.version.deposit
publishedVersion
en_US
ethz.identifier.nebis
006206116
ethz.publication.place
San Francisco, CA, USA
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::08816 - Kast, Peter (Tit.-Prof.)
en_US
ethz.leitzahl.certified
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::08816 - Kast, Peter (Tit.-Prof.)
ethz.date.deposited
2017-06-11T15:35:51Z
ethz.source
ECIT
ethz.identifier.importid
imp593652d3a428f81537
ethz.ecitpid
pub:151280
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2017-07-13T17:27:38Z
ethz.rosetta.lastUpdated
2018-11-01T10:32:38Z
ethz.rosetta.versionExported
true
ethz.COinS
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