Fig. 3: Chlorthalidone-binding site.

a Sliced view of one TMD subunit of chlorthalidone (CTN)-bound NCC_cryo. The surface representation of the protein is colored by electrostatic potential, in range of red (−5 kT  e⁻¹) to blue (+5 kT e⁻¹). The bound chlorthalidone molecule is shown as sticks. b Densities of chlorthalidone and nearby residues. hNCC and chlorthalidone are colored in tan and green, respectively. The contour levels of the left and right panels are 9.5 σ and 7.5 σ, respectively. c Chemical structure of chlorthalidone. The sulfamoyl and chlorine groups are colored red and green, respectively. The phthalimidine moiety is outlined with a red dashed oval, with the numbering system shown as blue numbers. d Chlorthalidone-NCC interactions. Hydrogen bonds are shown as dashed lines in yellow, and π–π stacking interactions in cyan. The color scheme is the same as in (b). e Effects of alanine substitutions of key CTN-interacting residues on hNCC sensitivity to chlorthalidone. Data are shown as mean ± SEM (n = 3 independent experiments). Data for WT are the same as in Fig. 1f. f Differential effects of N149A and N227A substitutions on hNCC sensitivities to several thiazide diuretics. The bar heights are best-fit values of IC₅₀ fold change relative to hNCC(WT) calculated from Figs. 2f (indapamide) and 3e (chlorthalidone) using the EC50 shift analysis in GraphPad Prism 10. For polythiazide, best-fit values were calculated from sensitivities reported in ref. ³⁶. The error bar depicts the standard error reported by GraphPad Prism 10, which serves as an indicator of the confidence of calculated IC₅₀ fold change.