Structural and Mechanistic Characterization of Mycobacterium tuberculosis TrxR Inhibition by Glutathione-Coated Gold Nanocluster

<i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) relies on the thioredoxin (Trx)-thioredoxin reductase (TrxR) system to maintain intracellular redox homeostasis and to support Trx-dependent DNA synthesis and repair, making TrxR a potential target for anti-tuberculosis therapy. Gold nanoclusters have been reported to inhibit human TrxR and suppress tumor growth, suggesting that gold-based nanomaterials can modulate TrxR activity. In this study, we report a previously uncharacterized oxidized crystal structure of <i>M. tuberculosis</i> TrxR containing two dimers in the asymmetric unit and use this structure to investigate inhibition by a glutathione-coated gold nanocluster (GSH-AuNC). Biolayer interferometry and enzymatic assays show that GSH-AuNC binds directly to <i>M. tuberculosis</i> TrxR and efficiently inhibits its catalytic activity at the purified enzyme level. Molecular dynamics simulations indicate that GSH-AuNC can occupy a surface pocket proximal to the active site, providing a plausible structural basis for enzyme engagement. AlphaFold3 modeling of the <i>M. tuberculosis</i> TrxR-Trx heterodimeric complex defines the interaction interface required for productive electron transfer and provides a structural hypothesis for how GSH-AuNC disrupts this process. Together, these results provide structural and mechanistic insights into the biochemical modulation of <i>M. tuberculosis</i> TrxR by GSH-AuNC, while the antimycobacterial activity of GSH-AuNC remains to be evaluated in future studies.