Folding stability at the anion-binding site distinguishes prestin from other SLC26 transporters as revealed by HDX-MS.

Abstract

The outer hair cell protein prestin (SLC26A5) responds to transmembrane voltage fluctuations by changing its cross-sectional area. This process, known as electromotility, is fundamental to mammalian cochlear amplification. Though prestin belongs to the SLC26 family of membrane transporters, it is the only member capable of displaying electromotility. Prestin's associated charge movement is driven by the helical dipole between TM3 and TM10, the charged residue R399, and the bound anion, but the relative roles remain to be determined. Here, we investigated these issues by interrogating the dynamics of prestin and SLC26A9, a typical SLC26 anion transporter, using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We show that the folding stability of the anion-binding site of prestin is at least 2.8 kcal/mol weaker than SLC26A9 while the remaining regions have similar stability. Anion binding to prestin reversibly folds the binding site by joining the TM3 and TM10 helices, ensuing allosteric modulation of the TMD dynamics. In contrast, anion binding minimally affects the stability of SLC26A9, including the TM3-TM10 dielectric gap. We find helix fraying at the binding site but cooperative unfolding for multiple peripheral helices, with potential structural and functional relevance. Our HDX-MS data not only validate recent cryo-EM structures, but also highlight the unique role of the bound anion driving the folding of prestin's anion-binding site. This phenomenon may have implications to prestin's voltage-sensing mechanism in the outer hair cells.