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 ␣-helical bundle and a short ␣-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 ␣-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 ␣-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. Among infectious human diseases, tuberculosis (TB)3 is the second greatest killer worldwide due to a single infectious agent, and remains a major challenge to human health care. In 2013, there were about 9 million new cases and 1.5 million deaths from TB, with an estimated one-third of the human population carrying a latent infection (1). First-line treatment for drug-susceptible TB requires the administration of a combination of four drugs during a period of 6 months: isoniazid, rifampicin, ethambutol, and pyrazinamide. Lengthy treatment regimens, unpleasant side effects, and patient noncompliance have provided conditions for the generation of multidrug-resistant and extensively drug-resistant cases of TB (2). Thus, the discovery and development of novel anti-TB drugs with bactericidal mechanisms different from those of currently available agents has become an urgent need. In that context, bedaquiline, a diarylquinoline that inhibits the c subunit of ATP synthase from Mycobacterium tuberculosis, has been recently approved by the Food and Drug Administration (FDA) for the treatment of multidrug-resistant TB in adults (3-5). Moreover, several candidate molecules are currently in preclinical studies, phase II and III clinical trials (6, 7). However, up to date, only a few drugs are capable of e...

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis

“…The crystal structure of the reduced form of Rv2466c has been solved recently, revealing a canonical thioredoxin fold with a Cys 19 -Pro 20 -Trp 21 -Cys 22 active-site motif, a typical feature of the thioredoxin superfamily of oxidoreductases (27). Rv2466c is a homodimer in which a ␤-strand is swapped between the thioredoxin fold domains of each subunit (28). As a consequence, the exposed face of the extended ␤-sheets forms a large, mostly hydrophobic groove flanked by an array of five ␣-helices on each side, where the active site is located (supplemental Fig.…”

“…tution of His 99 by serine leads to a remarkable decrease in activity (28). His 99 is proposed to stabilize the interface between the thioredoxin domain and the ␣-helical subdomain of Rv2466c and, as such, is directly involved in protein stability.…”

“…His 99 is proposed to stabilize the interface between the thioredoxin domain and the ␣-helical subdomain of Rv2466c and, as such, is directly involved in protein stability. This prediction comes from the observation that the H99S mutant undergoes a large structural change to adopt a conformation similar to that of the oxidized form of Rv2466c, indicative of a crucial structural role for His 99 (28).…”

“…To obtain a complemented strain, rv2466c was amplified with its upstream region (603 bp) using the primers RP1766 (5Ј-gatatccgccgttggagatcaccttc-3Ј/RP1767 (5Ј-gatatcccgacattgtgcccgaca-3Ј) and cloned into in an integrative plasmid based As previously demonstrated, Cys 19 and Cys 22 form a disulfide bond when under oxidative stress (17,28). 1, in the first chemical step, one molecule of mycothiol reacts with the nucleophilic Cys 19 , forming a mixed disulfide (R-S-SM).…”

Rv0579 Is Involved in the Resistance to the TP053 Antitubercular Prodrug

“…By microbiological, genetic, biochemical and crystallographic studies, its mechanism of activation was elucidated; TP053 is a prodrug activated by the reduced form of the DsbA-like mycoredoxin Mrx2 (encoded by Rv2466c gene), a mycothiol-dependent reductase with an unusual active-site motif CXXC (Albesa-Jové et al, 2014;Rosado et al, 2017). Mrx2 utilizes a chaperone-like mechanism of conformational changes, mainly in the CXXC active site motif, to recognize TP053 and promoting compound reduction (Albesa-Jové et al, 2015).…”