Show simple item record

dc.contributor.authorRoot, Katharina
dc.contributor.authorBarylyuk, Konstantin
dc.contributor.authorSchwab, Anatol
dc.contributor.authorThelemann, Jonas
dc.contributor.authorIllarionov, Boris
dc.contributor.authorGeist, Julie G
dc.contributor.authorGräwert, Tobias
dc.contributor.authorBacher, Adelbert
dc.contributor.authorFischer, Markus
dc.contributor.authorDiederich, François
dc.contributor.authorZenobi, Renato
dc.date.accessioned2018-11-17T00:31:57Z
dc.date.available2018-11-17T00:31:57Z
dc.date.issued2018-07-21
dc.identifier.issn2041-6520
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/285387
dc.description.abstractCharacterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana, an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of AtIspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of AtIspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of AtIspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.
dc.format.mediumElectronic-eCollection
dc.languageeng
dc.publisherRoyal Society of Chemistry (RSC)
dc.rightsAttribution-NonCommercial 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/
dc.titleAryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.
dc.typeArticle
prism.endingPage5986
prism.issueIdentifier27
prism.publicationDate2018
prism.publicationNameChem Sci
prism.startingPage5976
prism.volume9
dc.identifier.doi10.17863/CAM.32753
dcterms.dateAccepted2018-06-17
rioxxterms.versionofrecord10.1039/c8sc00814k
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2018-07
dc.contributor.orcidZenobi, Renato [0000-0001-5211-4358]
dc.identifier.eissn2041-6539
rioxxterms.typeJournal Article/Review
cam.issuedOnline2018-06-18


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record

Attribution-NonCommercial 4.0 International
Except where otherwise noted, this item's licence is described as Attribution-NonCommercial 4.0 International