Cryo-EM structures of thermostabilized prestin provide mechanistic insights underlying outer hair cell electromotility

Abstract

Outer hair cell elecromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized prestin (PresTS), complexed with chloride, sulfate, or salicylate at 3.52-3.63 Å resolutions. The central positively-charged cavity allows flexible binding of various anion species, which likely accounts for the known distinct modulations of nonlinear capacitance (NLC) by different anions. Comparisons of these PresTS structures with recent prestin structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insights into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange

“…The presumable location of a bound chloride ion is resolved in the structure of SLC26A6 at a low contour of the map (Figure 4B, Figure 2-figure supplement 2B). This position concurs with positions inferred from known structures of paralogs, which show binding of Cl - and HCO 3 to an equivalent location (Chi et al, 2020; Futamata et al, 2022; Ge et al, 2021; Liu et al, 2022) (Figure 4B, Figure 4-figure supplement 1). The anion binding site is placed between the opposed short helices α3 and α10, which partly span the membrane and whose N-termini are aligned to face each other in the center of the core domain (Figure 4A).…”

“…The presumable location of a bound chloride ion is resolved in the structure of SLC26A6 at a low contour of the map (Figure 4B, Figure 2-figure supplement 2B). This position concurs with positions inferred from known structures of paralogs, which show binding of Cland HCO3to an equivalent location (Chi et al, 2020;Futamata et al, 2022;Ge et al, 2021;Liu et al, 2022) (Figure 4B, Figure 4-figure supplement 1).…”

“…A dataset of the protein purified in the detergent glycol-diosgenin (GDN) at 3.3 Å was of high quality and allowed the unambiguous interpretation of the cryo-EM density by an atomic model (Figure 2A, Figure 2-figure supplements 1 and 2). In its organization, the SLC26A6 structure shows familiar features that have previously been observed in various eukaryotic family members of known structure including human and murine SLC26A9 (Chi et al, 2020; Walter et al, 2019), SLC26A5 (Bavi et al, 2021; Butan et al, 2022; Futamata et al, 2022; Ge et al, 2021), SLC26A4 (Liu et al, 2022) and the plant transporter AtSULTR4 (L. Wang et al, 2021) (Figure 2B, Figure 2-Figure Supplement 3).…”

“…The same site likely also provides a suitable environment for HCO3and oxalate, neither of which are substrates of SLC26A9 (Dorwart et al, 2007;Walter et al, 2019). A similar anion interaction observed in SLC26A6 is also found in the paralogs SLC26A4 (Pendrin) (Liu et al, 2022) and SLC26A5 (Prestin) (Futamata et al, 2022;Ge et al, 2021), which both share a comparable substrate preference. The similarity between the core domains of SLC26A6 and Prestin is remarkable in light of the altered function of the latter, which is no longer capable of transport and instead operates as motor protein in cochlear outer hair cells of mammals (Zheng et al, 2000).…”

Functional studies of deafness-associated pendrin and prestin variants

“…A recent cryo-EM study on mouse pendrin (Liu et al, 2023) revealed a homodimeric architecture that is similar to prestin (Bavi et al, 2021; Butan et al, 2022; Futamata et al, 2022; Ge et al, 2021) and other SLC26 proteins (Chi et al, 2020; Tippett et al, 2023; Walter et al, 2019). The transmembrane domain consists of the gate and the core domains, and the ion translocation pathway is located in between them ( Fig.…”