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Bedaquiline Binding at the Leading Site of Mycobacterium tuberculosis ATP Synthase Induces Distinct Structural and Dynamic Changes
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DOI：10.1002/jcb.70095.png)
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Abstract
En 中文
ATP synthase (ATPase) is a crucial molecular motor in Mycobacterium tuberculosis (Mtb), essential for energy production and oxygen-dependent pathogenesis. The enzyme consists of two distinct rotors: a membrane-embedded Fₒ unit and a cytosolic catalytic F1 unit, along with a stator, a central stalk, and a heterodimeric peripheral stalk (PS). As the F0 region hosts critical drug-binding pockets, it has gained significant interest. This study focuses on local structural dynamics at the leading site in the presence of bedaquiline (BDQ). All-atom molecular dynamics simulations were performed using GROMACS in a heterogeneous bilayer composed of phosphoinositol, phosphoethanolamine, phosphoglycerol, and cardiolipin (PI: PE: PG: CL) in a 32:42:4:50 ratio. The results revealed key interactions of BDQ with cL59, cF65, cE61, cA62, cI55, cI66, cG58, aI215, and aF219 at the a/c interface, consistent with energetically favored binding conformation. Quantitative lipid contact analysis revealed higher CL interactions with BDQ at leading site together with interfacial water molecules, whereas protein-lipid contacts based on only lipid headgroup (P-atoms) analysis remained independent of lipid abundance in the system. RMSD and RMSF revealed BDQ-induced fluctuations in the outer helix of subunit-c, while subunit-a remained comparatively more stable during the simulation. Distance analysis further indicated that the ligand remains confined within the binding region despite local flexibility. We further identified putative non-collinear proton channels, which showed no significant global perturbation upon BDQ binding. Residues aG195, aN105, aD220, aN190, aQ227, cE61 (inlet side), cE61 (outlet side), aE176, aE175, aA178, aK179, aS182, aY238, aQ110, aF192, aL122 form the two half channels in Mtb. The pooled water-count analysis for channels showed similar hydration levels in all simulated systems. We hypothesize that selective targeting of the leading pocket by newer drugs, in the presence of CL lipids, can modulate the proton inlet channel. The heterogeneous bilayer supports the structural and functional integrity of the membrane and ATPase complex. A CL-enriched membrane environment may provide a useful framework for investigating membrane-associated effects of BDQ and its analogs. Chain-wise analysis showed synchronous movement of the PS subunits and twisting of the δ-binding region, which may be perturbed in the presence of the F1 unit. The dynamics also revealed subunit-bδ involvement with the subunit-a at the leading pocket, a less studied PS and stator function. Identifying residue-specific interactions between PS could help to reveal its mechanical function. Together, these results provide complementary dynamic insights into BDQ at the leading pocket in a physiologically mimicked membrane environment and potentially support its relevance as a target site for the development of anti-TB compounds targeting ATPase.
Keywords:
heterogeneous bilayer
leading site
molecular dynamics simulation
multimeric protein assembly
proton-half channels
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