Impact of anionic lipids on the energy landscape of conformational transition in anion exchanger 1 (AE1)

Anion Exchanger 1 (AE1) is an elevator-type membrane transporter that plays a key role in erythrocytes by mediating the exchange of chloride and bicarbonate ions across the membrane, thus participating in acid-base homeostasis. While previous studies have provided structural insights into AE1 and its substrate binding, the conformational transitions and the role of lipid interactions remain elusive. In this study, we utilized cryo-electron microscopy (cryo-EM) to resolve three high resolution structures of distinct conformational states of AE1: two inward-facing (IF1 and IF2) and one outward-facing (OF). Furthermore, uptake assay revealed the modulatory effect of phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) lipids on AE1. Molecular dynamics (MD) simulations were conducted on these structures to capture anion binding and determine the anion binding sites in AE1. We then used a combination of advanced enhanced sampling techniques together with system-specific collective variables to study the OF⟺IF transition in AE1 and provided refined pathways for the process in three different systems: apo , HCO ₃ ⁻ -bound, and an AE1 system in which cryo-EM-determined interfacial PIP ₂ lipids had been removed. The refined pathways were then used to calculate the free energy of the OF⟺IF transition in AE1 under different conditions. The energies show how substrate binding reduces the transition barrier and, therefore, facilitates the transport. Furthermore, they clearly capture the inhibitory effect of PIP ₂ lipids at the dimer interface. Furthermore, the simulation results provide a molecular mechanism for this inhibitory effect. These results provide a molecular-level understanding of the mechanistic basis for ion transport in AE1 and the regulatory role of PIP ₂ on its function.