Uncovering structural and molecular dynamics of ESAT-6:B2M interaction: Asp53 of human B2-microglobulin is critical for the ESAT-6:B2M complexation

ESAT-6 is a small secreted protein of Mycobacterium tuberculosis involved in the ESAT-6 secretion system (ESX-1)–mediated virulence and pathogenesis. The protein interacts with b2M, causing downregulation of MHC class I Ag presentation, which could be one of the mechanisms by which it favors increased survival of the bacilli inside the host. In an earlier study, we have shown that the C-terminal region of ESAT-6 is crucial for its interaction with b2M. However, the interface of b2M involved in interaction with ESAT-6 and detailed physicochemical changes associated with ESAT-6:b2M complexation are not fully defined. In this study, using computational and site-directed mutagenesis studies, we demonstrate the presence of strong noncovalent hydrophobic interactions between ESAT-6 and b2M in addition to the vital hydrogen bonding between the aspartate residue (Asp53) of b2M and methionine (Met93) of ESAT-6. Docking-based high-throughput virtual screening followed by 16-point screening on microscale thermophoresis resulted in the identification of two potent inhibitors (SM09 and SM15) that mask the critical Met93 residue of ESAT-6 that is required for ESAT-6:b2M interaction and could rescue cell surface expression of b2M and HLA in human macrophages as well as MHC class I Ag presentation suppressed by ESAT-6 in peritoneal macrophages isolated from C57BL/6 mice. Both SM09 and SM15 significantly inhibited intracellular survival of M. tuberculosis in human macrophages. Further, we characterized the physicochemical properties involved in the ESAT-6:b2M complexation, which may help in understanding host–pathogen interactions.