Abstract
Prestin, a member of the SLC26A family, is essential for the electromotility of mammalian outer hair cells, converting voltage changes into mechanical work. In contrast, nonmammalian orthologues function as anion transporters. To investigate the molecular and structural basis of this functional divergence, we performed ancestral sequence reconstruction (ASR) of prestin across vertebrates, followed by structural modeling using AlphaFold2-multimer and molecular dynamics simulations. We identified more than 200 amino acid substitutions along the lineage that lead to placental mammals, with early substitutions concentrated in the transmembrane domain (TMD) and late substitutions clustering in the STAS domain, particularly in the intervening sequence (IVS). Structural modeling and simulation revealed that early substitutions modulate protein-lipid interactions and interhelical contacts. In placental mammals, the IVS-loop adopts a distinct conformation that places a negatively charged patch near the chloride access pathway, potentially affecting the ion dynamics and voltage responsiveness. These structural transitions occurred without major rearrangements of the global fold of prestin, supporting a notion in which the novel function evolved through distributed substitutions within a conserved scaffold. Our findings illustrate how molecular exaptation, and incremental structural remodeling enabled the repurposing of an ancestral anion transporter into a voltage-sensitive area-motor, providing a framework for understanding the molecular evolution of complex biophysical traits central to auditory neuroscience.