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The redox state regulates the conformation of Rv2466c to activate the antitubercular prodrug TP053

DOI: 10.1074/jbc.M115.677039 DOI Help
PMID: 26546681 PMID Help

Authors: David Albesa-jove (Unit of Biophysics) , Natalia Comino (Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea, Spain) , Montse Tersa (Unit of Biophysics) , Elisabeth Mohorko (ETH Zurich, Switzerland) , Saioa Urresti (Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea, Spain) , Elisa Dainese (University of Padova, Italy) , Laurent R. Chiarelli (University of Pavia, Italy) , Maria Rosalia Pasca (University of Pavia, Italy) , Riccardo Manganelli (University of Padova, Italy) , Vadim Makarov (Russian Academy of Science, Russian Federation) , Giovanna Riccardi (University of Pavia, Italy) , Dmitri I. Svergun (EMBL Hamburg, Germany) , Rudi Glockshuber (ETH Zurich, Switzerland) , Marcelo Guerin (Unit of Biophysics)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: November 2015
Diamond Proposal Number(s): 8302 , 10130

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 Cys19-Pro20-Trp21-Cys22 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.

Journal Keywords: Conformational Change Enzyme Mycobacterium Tuberculosis Oxidation-Reduction (Redox) Small-Angle X-Ray Scattering (Saxs) Thioredoxin X-Ray Crystallography

Subject Areas: Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography

Other Facilities: EMBL; PETRA; DESY; SOLEIL