Structural Characterization of Mycobacterium tuberculosis IspQ Reveals Membrane-Dependent Stabilization of Its Conserved Electron-Transfer Cofactor Network

Abstract

Mycobacterium tuberculosis persistence in granulomas depends on lipid catabolism through the β-oxidation-coupled respiratory pathway, which requires the essential membrane-associated protein IspQ, encoded by Rv0338c. Although IspQ is a genetically and chemically validated drug target, its structural organization and membrane-dependent function remain unknown. Using AlphaFold3 structure prediction, cofactor mapping, evolutionary conservation analysis, and atomistic molecular dynamics simulations, we show that IspQ adopts an EtfD-like fold containing four iron–sulfur clusters, a heme b, and a menaquinone-binding pocket, mapped from the experimental EtfD structure, to form a continuous electron-transfer relay, with all 25 coordinating residues corresponding one-to-one with their experimentally assigned counterparts. Simulations across three independent replicates in each of three membrane systems reveal that the native mycobacterial membrane produces the lowest global conformational deviation, reduces the flexibility of cofactor-coordinating residues by 24% relative to a simple bilayer, and maintains the catalytic menaquinone-binding heme pocket in a geometry closest to that of the cofactor-loaded experimental structure. Native lipids form persistent interactions with IspQ, defining conserved protein–lipid interfaces. These findings establish a structural and dynamic framework for IspQ, reveal membrane-dependent modulation of its electron-transfer machinery, and identify these interfaces as candidate sites for structure-guided antitubercular drug discovery.