Distinct HDX response of prestin and SLC26A9 to Cl- binding.

(A) HDX data analysis to obtain (B) and (C). One example peptide is shown in cases where HDX becomes faster or slower in Hepes (the apo state) compared to in Cl-. Deuteration levels are obtained from the mass spectra. Here spectra for the undeuterated peptide (grey) and after 5 min HDX labeling in Cl- (black) and Hepes (green) are shown as an example. The resulting deuterium uptake plots are used to generate the differential deuteration heatmaps in (B). Changes in free energy of unfolding (ΔΔG) in (C) are calculated after fitting the data with a stretched exponential (Materials and Methods)17. (B) Heatmaps showing the difference in deuteration levels at each labeling time for all TMD peptides of prestin and SLC26A9 measured in Hepes compared to Cl-. Peptide sequences corresponding to peptide indexes can be found in Fig. S9 & Fig. S10. (C) The ΔΔGs in Hepes compared to Cl- for full-length prestin and SLC26A9 mapped onto the structure (PDB 7S8X and 6RTC). Red and blue indicate increased and decreased stability upon Cl- binding, respectively. Following regions of the left subunits are shown as low transparency to highlight the binding site – prestin: TM5 and TM12-14; SLC26A9: TM5 and TM13-14. Regions with no fitting results are in grey.

The anion-binding pockets for prestin and SLC26A9 exhibit distinct stability changes upon Cl- binding, albeit highly conserved.

(A-B) Cl- binding stabilizes prestin’s anion-binding pocket (A) but mildly affects SLC26A9’s (B). The structure shows the anion-binding pocket (TM1, TM3, and TM10) with the putative position of the bound Cl-. Colored regions correspond to peptides whose deuterium uptake plots are shown (prolines are colored in grey) when the protein is in Cl- (black) and in Hepes (red). Grey dashed lines indicate deuteration levels in the full-D control. Data from two and three biological replicates are shown for prestin in Cl- and Hepes, respectively. Data from three technical replicates are shown for SLC26A9. Replicates are shown as circles, triangles, and squares. Some replicates are superimposable and hence not observable. The symbols (* and #) in (A.b) denote data points used in Fig. 3B. (C) Sequence alignment using Clustal Omega of prestin and close SLC26 transporters across species for the anion-binding pocket. Shades of blue indicate degree of conservation.

Anion binding folds and stabilizes prestin’s binding site.

(A) Deuterium uptake plots for the N-terminus of TM3 (Peptide134-140) measured in the absence and presence of 4 M urea, in a background of (Left) Cl- and (Right) Hepes. Replicates (circles, triangles, and squares): 2 in Cl-, 3 in Hepes, 2 in Hepes with urea, biological. Grey dashed curves represent deuterium uptake with kchem34,35, normalized with the back-exchange level. (B) Deuteration levels for (Left) the N-terminus of TM3 (Peptide134-142) in three biological replicates and for (Right) TM1 (Peptide84-101) after 5 min labeling upon titrating Cl- to apo state of prestin. Dashed lines indicate deuteration levels at t = 5 min (* and # for apo and Cl--bound states, respectively) taken from Fig. 2A.b and Fig. S9.11. Residues in grey denoted in the peptide sequence do not contribute to the deuterium uptake curve.

Prestin’s dynamics are regulated by anions of varying identities.

(A) Heatmaps showing the difference in deuteration levels at each labeling time for all TMD peptides measured in SO42- or salicylate compared to Cl-. Peptide sequences corresponding to peptide indexes can be found in Fig. S9. (B) The structure shows the anion-binding pocket with the putative position of the bound Cl-. The pink mesh highlights the region with the greatest HDX response to binding to various anions. Colored regions correspond to peptides whose deuterium uptake plots are shown (prolines are colored in grey) when the protein is in Cl- (black, two biological replicates shown in circles and triangles), SO42- (green), and salicylate (blue). Grey dashed lines indicate deuteration levels in the full-D control.

Helix folding cooperativity and the proposed mechanism for prestin’s electromotility.

(A) Left: Deuterium buildup curves for (i) the N-terminal TM3 (Peptide134-140) and (ii) the intracellular portion of TM6 (Peptide273-282) in Cl- depicting helix fraying and mild cooperativity, respectively. Circles: experimental deuteration levels, normalized with in- and back-exchange levels. Grey dashed curves: hypothetical intrinsic uptake curves (PF = 1). On the top shows individual exponentials whose sum is fitted to the experimental values and plotted on the main buildup curves. Residues in grey denoted in the peptide sequence do not contribute to the deuterium uptake curve. Upper right: χ2 and the relative error as the number of fit exponentials increases, used to assess the quality of fit. Lower right: Models and free energy surface of unfolding illustrating the difference between (i) fraying and (ii) mild cooperativity. (B) Mechanism for prestin’s conformational transition from the expanded to the contracted state regulated by the anion concentration. Green rectangles and curved lines: folded and unfolded fractions, respectively, of TM3 and TM10. Blue filled circle: R399. Blue dashed circle: partial positive charges from TM3 and TM10 helical dipoles. Red filled circle: anions, with the size of the circle depicting anion concentrations. Black arrows: prestin’s conformational change.