Peripheral Stalk-Associated Membrane Perturbations around Mycobacterium tuberculosis ATP Synthase

Abstract The mechanism of ATP synthase (ATPase) has been extensively investigated, yet its structural complexity challenges understanding membrane-protein dynamics at the molecular scale. In this study, we prepared a heterogeneous bilayer based on experimental composition, comprising phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL) at 42, 32, 4, and 50 molecules per leaflet, respectively, representing a simplified model of the Mycobacterium inner membrane. We simulated three systems using a reduced-complexity model: Mycobacterium tuberculosis(Mtb) ATPase with full peripheral stalks (PS; system S1), a trimmed PS variant (S2), and a protein-free bilayer control (S3). We prepared and simulated the systems for 300 ns, with independent replicas for the protein-containing systems. Bilayer properties, like area per lipid (APL), bilayer thickness, deuterium order parameters SCD, density distributions, lateral diffusion coefficients, and protein–lipid contact frequencies, were analyzed using block averaging and probability distributions. Comparative analysis revealed that ATPase architecture selectively remodels the local membrane environment and induces leaflet-specific membrane organization while maintaining global bilayer stability. The full-PS S1 system exhibited increased interfacial water density, tighter annular lipid packing, and distinct APL, highlighting local changes. These ATPase-associated membrane perturbations differed in S2 and were absent in the protein-free S3 system. Both PS and the heterogeneous lipid composition contribute to lipid redistribution and modulation of membrane parameters, including APL (S2 < S1 < S3; 0.54, 0.55, and 0.60 nm2), thickness (S1 < S2 < S3; 4.052 < 4.085 < 4.106 nm), and density profiles. Lateral diffusion followed the trend S3 < S1 < S2, with bilayer composition regulating dynamics despite larger APL in S3. The normalized protein–lipid contact frequencies showed comparable contacts for CL and PI. Lipid component density profiles further suggested that CL may exhibit positional organization around the transmembrane Fo unit, consistent with its distinct tetra-acyl-chain and headgroup structure. These findings provide atomistic insights into the reciprocal coupling between ATPase structure and heterogeneous membrane organization, highlighting the combined influence of lipid composition and PS-associated structural effects on the ATPase-membrane interface, its function and disease mechanisms.