Extended Data Fig. 9: Molecular docking simulations show stepwise reductions in ouabain binding to Na⁺/K⁺-ATPases with monarch lineage substitutions in ATPα.

The ouabain binding pocket structure obtained from molecular docking simulations for each Na⁺/K⁺-ATPase with mutated ATPα. The mutated residues are shown in sticks and are labelled. The H1–H2 loop of ATPα is shown in blue. The extracellular region of the α-subunit is removed for simplicity. For the wild-type (QAN) ATPase, ouabain is shown in its co-crystal structure coordinates (white, transparent) together with its best-docked position. For all other ATPases only the best-docked positions (closest to the co-crystal structure) are shown together with ouabain’s docking position for the wild-type ATPase (dark grey). The triple-mutated VSH ATPase has two distinct docking scores: one is similar to the docking energy for the wild-type ATPase and the other has the lowest binding energy compared to all other mutated ATPases. The potential existence of both states might be related to a trend of reduced bang sensitivity for this genotype compared to some of the single-mutant genotypes. A119 is not directly part of the ouabain binding pocket, and therefore, A119S alone does not change ouabain binding. Although the consequences of A119S are relatively subtle, the mutation may disrupt the local hydrogen bonding network and cause structural or dynamic changes in the loop or in its vicinity.