The exceptional hearing sensitivity of mammals depends on cochlear amplification, a process driven by the rapid electromotility of outer hair cells. At the molecular level, electromotility is driven by prestin, a membrane motor protein that undergoes voltage-driven in-plane area expansion and contraction across acoustic frequencies. Although the electromotile function of prestin has extensively been studied functionally, the structural details that underlie this function remain unclear. In light of this, the overarching aim of my thesis is to deepen our understanding of the molecular mechanisms by which prestin drives electromotility, and how its motor function evolved from an ancestral transport mechanism. In the first part of the thesis, we determine cryo-EM structures of mammalian motor and nonmammalian transporter prestin and provide molecular insights into how these functions are regulated by the membrane environment. Our results suggest that a key distinction between orthologs is how they link their conformational states to the membrane, providing a molecular rationale for how a transporter scaffold was repurposed into a highly efficient membrane motor. In the second part of the thesis, we further examine the evolutionary trajectory of this functional distinction using a combination of cryo-EM, structure-guided mutagenesis, and functional assays. Our results argue that the core voltage-sensing mechanism is qualitatively conserved across orthologs, and consistent with the first part of the thesis, implicate changes at the protein-lipid interface as an important aspect of the functional transition. In the third part, we examine whether prestin forms stable interactions with other proteins to perform its function in outer hair cells. Proteomics of prestin purified from native outer hair cells provides no evidence for stable protein-protein interactions, consistent with the idea that prestin functions as a self-sufficient membrane motor. Together, the results from this thesis illuminate the structural and evolutionary principles by which prestin operates as a membrane motor and shed new light into the molecular basis of cochlear amplification in mammals.
