Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.
View / Open Files
Authors
Root, Katharina
Barylyuk, Konstantin
Schwab, Anatol
Thelemann, Jonas
Illarionov, Boris
Geist, Julie G
Gräwert, Tobias
Bacher, Adelbert
Fischer, Markus
Diederich, François
Publication Date
2018-07-21Journal Title
Chem Sci
ISSN
2041-6520
Publisher
Royal Society of Chemistry (RSC)
Volume
9
Issue
27
Pages
5976-5986
Language
eng
Type
Article
Physical Medium
Electronic-eCollection
Metadata
Show full item recordCitation
Root, K., Barylyuk, K., Schwab, A., Thelemann, J., Illarionov, B., Geist, J. G., Gräwert, T., et al. (2018). Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.. Chem Sci, 9 (27), 5976-5986. https://doi.org/10.1039/c8sc00814k
Abstract
Characterizing 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.
Identifiers
External DOI: https://doi.org/10.1039/c8sc00814k
This record's URL: https://www.repository.cam.ac.uk/handle/1810/285387
Rights
Attribution-NonCommercial 4.0 International
Licence URL: https://creativecommons.org/licenses/by-nc/4.0/
Statistics
Total file downloads (since January 2020). For more information on metrics see the
IRUS guide.