title: The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.
abstract: Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.
authors.affiliation: From the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain, the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain, the IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Albesa-Jové
authors.affiliation: From the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain, the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Comino
authors.affiliation: From the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain, the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain.
authors.forename: Montse
authors.initials: M
authors.lastname: Tersa
authors.affiliation: the Institute for Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mohorko
authors.affiliation: From the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain, the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain.
authors.forename: Saioa
authors.initials: S
authors.lastname: Urresti
authors.affiliation: the Departments of Molecular Medicine and.
authors.forename: Elisa
authors.initials: E
authors.lastname: Dainese
authors.affiliation: Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, 27100 Pavia, Italy.
authors.forename: Laurent R
authors.initials: LR
authors.lastname: Chiarelli
authors.affiliation: Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, 27100 Pavia, Italy.
authors.forename: Maria Rosalia
authors.initials: MR
authors.lastname: Pasca
authors.affiliation: the Departments of Molecular Medicine and.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Manganelli
authors.affiliation: the A. N. Bakh Institute of Biochemistry, Russian Academy of Science, 119071 Moscow, Russia, and.
authors.forename: Vadim
authors.initials: V
authors.lastname: Makarov
authors.affiliation: Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, 27100 Pavia, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Riccardi
authors.affiliation: the European Molecular Biology Laboratory, Hamburg Outstation, c/o Deutsches Elektronen-Synchrotron (DESY), Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: the Institute for Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Rudi
authors.initials: R
authors.lastname: Glockshuber
authors.affiliation: From the Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas, Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain, the Departamento de Bioquímica, Universidad del País Vasco, Leioa, Bizkaia 48940, Spain, the IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain, mrcguerin@gmail.com.
authors.forename: Marcelo E
authors.initials: ME
authors.lastname: Guerin
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D011355:Prodrugs
keywords: Mycobacterium tuberculosis; conformational change; enzyme; oxidation-reduction (redox); small-angle x-ray scattering (SAXS); thioredoxin; x-ray crystallography