Structure bioinformatics of eight human ATP synthase F0 subunits and their AlphaFold3-predicted reduced-hydrophobicity QTY analogues.

Human mitochondrial ATP synthase is an essential rotary motor enzyme that produces most of the cellular ATP through oxidative phosphorylation. Its membrane-embedded F0 sector contains highly hydrophobic transmembrane subunits that are challenging to study in aqueous environments without detergents. We applied the QTY code to eight human ATP synthase F0 subunits: ATP6, ATP8, ATPK, ATP68, ATPMK, AT5G1, AT5G2, and AT5G3. Hydrophobic amino acids leucine (L), isoleucine (I), valine (V), and phenylalanine (F) in transmembrane regions were systematically replaced with hydrophilic glutamine (Q), threonine (T), and tyrosine (Y). Four native subunits with available CryoEM structures from human ATP synthase (PDB: 8H9S) were superposed with their AlphaFold3-predicted QTY analogues. For subunits without complete CryoEM structures, AlphaFold3-predicted native structures were superposed with their AlphaFold3-predicted QTY analogues. Additional structural analyses were included, and key structural and functional regions, including ATP68 and the ATP6-c-ring interface, were modeled. The native ATP synthase F0 subunits superposed well with their respective QTY analogues. For the CryoEM-native comparisons, RMSD values ranged from 0.565 Å to 6.952 Å. For the AlphaFold3-native comparisons of subunits without CryoEM structures, RMSD values ranged from 0.204Å to 0.297Å. Considering all eight subunits, despite substantial QTY substitutions in the transmembrane regions, ranging from 38.89% to 50.79%, the QTY analogues retained similar overall folds, molecular weights, and isoelectric points. Hydrophobic surface analysis showed that the QTY analogues had reduced hydrophobic surfaces compared with their native counterparts. These structural bioinformatics studies suggest that the QTY code is applicable to ATP synthase F0 subunits to generate more hydrophilic, water-soluble analogues while preserving overall structural similarity. These results extend the application of the QTY code to the membrane-embedded F0 sector of ATP synthase and provide a foundation for future experimental studies for these QTY analogues expressed, purified, and evaluated for self-assembly.