Disruption of redox catalytic functions of peroxiredoxin-thioredoxin complex in Mycobacterium tuberculosis H37Rv using small interface binding molecules
Graphical abstract
Introduction
Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis, which had a mortality rate of 1.5 million people in 2014 (WHO Global Tuberculosis Report, 2015). It is estimated that one third of the world’s population is infected with the latent form of M. tuberculosis (Jasmer et al., 2002). The ability of M. tuberculosis to escape the host immune surveillance mechanism is primarily due to their ability to reside in the alveolar phagocytes which confer them resistance against oxidative killing and survivability (Akif et al., 2008). Therefore, disrupting this pathogen defense ability could possibly kill or inhibit their growth, and, hence proteins involved in this defense functions can be pursued as antimycobacterial drug targets. The mechanism by which the host phagocytes attempt to kill the invading pathogen is via production of reactive oxygen and nitrogen species including superoxides and hydrogen peroxides which are toxic to microbe (Shinnick et al., 1995). One of the mechanisms by which M. tuberculosis resist oxidative killing is via the same process which eukaryotic cells use to combat oxidative stress using the thiol reductants of the thioredoxin (Trx) system and peroxiredoxin (Prx) system. In other prokaryotic organisms, the Trx system keeps cellular proteins in a reduced state; along with glutathione (mycothiol is used in M. tuberculosis) (Holmgren, 2000, Arnér and Holmgren, 2000).
Prx is an essential thiol and non-heme peroxidase that functions together with Trx, sometimes with glutaredoxin-glutathion or other reductant as cyclophilin, to reduce peroxides at low concentrations of substrates (Noguera-Mazon et al., 2006). It also reduces peroxinitrite under certain conditions (Bryk et al., 2000). The Prx family includes six isoforms in mammals (Prx I to VI) (Seo et al., 2000), five in Saccharomyces cerevisiae (Park et al., 2000) and Drosophila melanogaster (Radyuk et al., 2001), upto 10 in plants (Rouhier and Jacquot, 2002, Dietz, 2003). It can be typically classified into four types: 1-Cys, atypical, 2-Cys and 3-Cys according to its enzymatic mechanism and the cysteine set involved in their catalytic cycle. Fig. 1 explains catalytic cycle of Prx involving atypical 2-cys mechanism in M. tuberculosis. The catalytic cycle of Prx involves two phases- a) oxidation via formation of disulfide bridges and b) reduction of disulfide bridges by a dithiol reductase such as Trx. The oxidized Trx is restored back to the reduced state by thioredoxin reductases (TrxR).
M. tuberculosis possesses an enzyme, AhpC (Alkyl Hyderoperoxide reductase) which belongs to 2-Cys class of Prx family. Although it is considered as a typical 2-Cys peroxiredoxin, it differs in a number of ways from other members of the family. First, it possess three cysteine residues instead of two, which are directly involved in catalysis (Chauhan and Mande, 2002) including the conserved peroxidasic cysteine (Sp) Cys61, the putative resolving cysteine (SR) Cys174 and a third catalytic cysteine, cys176 whose function is not clear (Chauhan and Mande, 2002; Koshkin et al., 2004). In M. tuberculosis AhpC is reduced by thioredoxin system consisting of thioredoxin C (TrxC) and thioredoxin reductase (TrxR) (Jaeger et al., 2004). In mycobacteria, AhpC not only detoxify hydroperoxides but also offers protection against reactive nitrogen intermediates (Chen et al., 1998, Master et al., 2002). The thioredoxin system in M. tuberculosis consists of three thioredoxin (TrxA, TrxB, TrxC) and one thioredoxin reductase (TrxR) (Akif et al., 2008, Akif et al., 2004, Hall et al., 2006). Thioredoxins are ubiquitous enzymes (Holmgren, 1985) that possess a conserved catalytic motif WCXXC and have a low molecular weight of about 12 kDa (Martin, 1995). They catalyze thiol-disulfide exchange reactions using redox active cysteine thiols to reduce oxidized disulfide cysteines of proteins including peroxiredoxins (Gleason and Holmgren, 1988, Ortenberg et al., 2004). The oxidized thioredoxins are then reduced by TrxR in an NADPH-dependent reaction via redox active cysteine or selenocysteine thiols depending on the organism (Williams et al., 2000, Zhong et al., 2000). This is achieved by formation of a heterodimer between Trx and TrxR which allows transfer of electrons to the Trx in a disulfide exchange reaction (Zhong et al., 2000). There are several challenges in designing small therapeutic compounds which modulate protein interactions (Fuller et al., 2009, Arkin and Wells, 2004, Wells and McClendon, 2007) but over the past, many workers have reported effective protein- protein interface binding modulators using molecular docking approach (Li et al., 1997, Fujii et al., 2003, Koehler et al., 2004, Gao et al., 2004, Nikolovska-Coleska et al., 2004).
There are large evidences that plant Trx specifically targets their interacting partners, Prx in vivo (Vignols et al., 2005, Verdoucq et al., 1999, Mouaheb et al., 1998) but the crystal structures of their complexes has not been reported to date. The present study aims to identify potential interface binding molecules which can stabilize interaction between Prx and Trx in M. tuberculosis which in turn can lock the protein complex in an inactive state thereby disrupting the redox catalytic functions of Prx and Trx. The work plan involved in this study is schematically represented in Fig. 2. In this computational study, we have modeled Prx-Trx complex of M. tuberculosis through protein–protein interactive approach and have characterized some salient features of protein–protein interface such as interface residues, contact area and solvent accessibility in this complex. This characteristic feature of interface region was utilized in virtual screening of possible drug-like compounds which can selectively bind to the Prx-Trx protein interface regions. Further, to gain insights into the binding mode of ligand with the complex, we conducted molecular dynamics simulation and principal component analysis (PCA) of both apo and ligand bound states.
Section snippets
Protein- protein interaction analysis
The primary sequences of Prx and Trx were retrieved from UniProt database (http://www.uniprot.org/) via their accession IDs P9WQB7 and P9WG67 respectively. STRING (The Search Tool for the Retrieval of Interacting Genes/Proteins) v10 database (Szklarczyk et al., 2015) was used to analyze the interaction between Prx and Trx. The active prediction methods in STRING include Neighbourhood, Gene fusion, Co-occurrence, Co-expression, Experiments, Databases and Textmining. The required confidence score
Protein-protein interaction analysis
Both peroxiredoxin and thioredoxin interact with each other with a high confidence score of 0.991, which is the total contribution from prediction methods such as neighbourhood, coexpression, experiments, databases and textmining (Fig. 3A and B). This confirms the possible interaction between Prx and Trx in M. tuberculosis.
Protein-protein docking was performed using HADDOCK web server. One hundred sixty eight structures were generated, which were grouped in 10 clusters and ranked according to
Conclusion
Peroxiredoxin (Prx) and thioredoxin (Trx) are two key enzymes ubiquitously present in many organisms including M. tuberculosis. M. tuberculosis employs these two key enzymes to combat the oxidative stress due to production of superoxides and reactive nitrogen intermediates in the host. This is possibly achieved through a critical interaction between Prx and Trx and thus disrupting this essential interaction could be envisaged as a novel endeavour to tackle with the pathogen. Therefore, we
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
The authors are thankful to the Department of Biotechnology, Government of India (GOI) for providing the support for this study (sanction order No. BT/326/NE/TBP/2012). The author ABG is thankful to the Department of Biotechnology, GOI for providing DBT-Junior Research Fellowship (DBT/JRF/13/AL/4294104).
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