Mycobacterium tuberculosis low molecular weight T-cell antigen Mtb8.4 has heme-binding and fiber-forming properties

Mtb8.4, a secretory T-cell antigen of Mycobacterium tuberculosis, is important for providing an antigen-specific immune response. In this study, we showed Mtb8.4 to have both heme-binding and fibril-forming properties, using experimental and in silico methods. High absorbance at 410 nm and interaction with hemin-agarose demonstrated its heme-binding nature. Titration of Mtb8.4 with heme resulted in 1 : 1 stoichiometry. The heme-binding pocket in Mtb8.4 was identified by molecular modeling, and binding residues were predicted using molecular docking. The molecular dynamics simulations of apo- and heme-bound Mtb8.4 confirmed that the heme group forms a stable complex. Transmission electron microscopy analyses and dye-binding assays showed that Mtb8.4 forms fibers. Computational studies predicted that the C-terminal sequence (⁹³ AAQYIGLVESV¹⁰³ ) is important for forming fibers. In silico analyses further anticipated the probable epitope (⁸² AMAAQLQAV⁹⁰ ) of Mtb8.4. The fiber-forming properties of Mtb8.4 could be advantageous from a vaccine perspective for aggregate/fibril-based vaccine delivery or it might influence the epitope presentation of Mtb8.4.