Molecular mechanism of prestin electromotive signal amplification

Abstract

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

Keywords: cochlear amplification; cryo-EM; electromotility; hearing; intrinsic voltage sensor; mechanotransduction; membrane protein; outer hair cells; prestin; protein lipid interaction.

Figure 1.. Function and cryo-EM structure of human prestin.

A. Non-linear capacitance measurement of HEK293 cells expressing human prestin. B. Schematic of the prestin secondary structure features. The features are colored based on panel D. Chloride is shown as a green dot. Neutral, positively and negatively charged residues that affect the prestin NLC in previous studies are shown as yellow, blue and red dots, respectively. C. Cryo-EM maps of prestin bound with chloride (Pres-Cl). The two protomers are colored red and blue. Resolved lipid molecules are shown in a transparent yellow surface. Densities representing cholesterol molecules are colored in purple. D. Structural model of Pres-Cl shown in ribbon representation. Key domains are colored in a single protomer. See also Figures S1–S7

Figure 2.. Anion binding pocket.

A-B. Electrostatic potential surfaces of the core domain of the Pres-Cl (A) and Pres-Sal (B) structures. Cl⁻ is shown as a green sphere. Salicylate is shown as sticks. C-D. Cartoon representation of the anion binding pocket of the Pres-Cl (C) and the Pres-Sal (D) structures. The core domain from one TMD is shown in each panel. Helices flanking the anion binding site are colored in purple. The cryo-EM densities of Cl⁻ or salicylate anion are shown as mesh. E. Details of the Cl⁻ binding pocket of Pres-Cl. Left panel, interactions of R399 proximal to the pocket. Right panel, interactions between the Cl⁻ ion and surrounding hydrophilic residues. Blue arrows indicate the helical dipoles of TMs 3 and 10. F. Details of the salicylate binding pocket of Pres-Sal. G. Computational model of the prestin F101Y mutant and binding of 5-methylsalicylate. H. Measurements of changes in NLC amplitude upon application of 10mM of either salicylate or 5-methylsalicylate in cells transfected with WT prestin or the F101Y mutant. See also Figures S1, S2, S4, S8, S9

Figure 3.. Chloride and salicylate stabilize distinct TMD conformations.

A-C. Conformational differences in the TMDs of the Pres-Cl and Pres-Sal complexes. TMDs from one protomer of Pres-Cl and Pres-Sal were superimposed based on the core domain. (A) The gate domain, shown as cartoon, has a 4.6° rotation around an axis approximately parallel to the membrane (labeled in red). The core domains are shown as grey surfaces. (B) A 90° rotated view of (A) showing the separation of the core and gate domains in Pres-Sal relative to Pres-Cl. (C) Distance measurements between the anion binding site, defined by the center of mass of residues 396–398, and the center of mass of each helix in the gate domain of Pres-Cl and Pres-Sal. D-F. Structural comparisons of prestin structures with homologous proteins. (D) Structural comparison of Pres-Cl, Pres-Sal, Pres-sulfate, the inward-facing state of SLC26A9 (PDB:7CH1) and the outward-facing state of AE1 (PDB: 4YZF). The structures are superimposed based on the gate domain. (E) View of TMs 3 and 10 that form the anion binding pocket. A key anion binding residue S398 in prestin and its equivalent residues V729 in AE1 and S392 in SLC26A9 are labeled. (F) Schematic representation showing how the different prestin conformational states correspond to conformations of homologous transporter proteins. See also Figures S8, S10, S11

Figure 4.. Prestin adopts an expanded conformation in the absence of chloride.

A-B. Tube representation of Pres-Sal (colored blue and red for each chain) or Pres-Cl (grey), superimposed based on the STAS domain. C. Cross sectional area of the prestin dimer in Pres-Sal and Pres-Cl, measured along the two-fold axis using a thresholded Gaussian map (Supplemental Information). D. Conformational displacements of the anion binding site relative to the STAS domain between Pres-Sal (colored) and Pres-Cl (grey). Cα atoms of residues used to measure displacements are indicated by spheres. E. Schematic representation of the displacement of the four residues highlighted in panel D. Dashed lines indicate distances between the Cα atoms within each state. Dashed arrows indicate the absolute distance between Cα atoms of each residue between Pres-Sal and Pres-Cl. Solid arrows indicate the component of the same distance along the two-fold axis. See also Figures S11, 12, Movie S1

Figure 5.. Prestin deforms the lipid bilayer.

A-B. Pres-Cl, solved in the presence of GDN, shown in surface representation where the surface is colored based on lipophilicity, from gold (lipophilic) to blue (hydrophilic). Modeled lipids from the upper leaflet are shown as green sticks and lipids from the lower leaflet are shown as magenta sticks. The position of the chloride atom is indicated using a blue mark. C. View of the TMD of a single prestin protomer together with modeled lipids and their corresponding cryo-EM density (blue mesh). The density modeled as cholesterol is substantially flatter than other lipid densities. D-E. Heatmaps of the average lipid headgroup height during a 1.5-μs MD simulation in the lower leaflet (D) and upper leaflet (E). See also Figures S3, S4

Figure 6.. Mechanism of prestin.

Left, prestin is in a contracted state in the presence of Cl⁻. A saddle-shaped membrane is formed around prestin. Two cholesterol molecules are bound in the center gap between the TMDs and the lower point of the upper membrane. Middle, absence of Cl⁻ induces both the expansion of the TMDs and the elevation of the core domain including the anion-binding pocket. Right, salicylate locks prestin in the expanded state. The binding of salicylate does not permit contraction, thus inhibiting the eM function of prestin.