Structural insights into the gating mechanism of human SLC26A9 mediated by its C-terminal sequence

Abstract

The human SLC26 transporter family exhibits various transport characteristics, and family member SLC26A9 performs multiple roles, including acting as Cl^-/HCO₃^- exchangers, Cl^- channels, and Na⁺ transporters. Some mutations of SLC26A9 are correlated with abnormalities in respiration and digestion systems. As a potential target colocalizing with CFTR in cystic fibrosis patients, SLC26A9 is of great value in drug development. Here, we present a cryo-EM structure of the human SLC26A9 dimer at 2.6 Å resolution. A segment at the C-terminal end is bound to the entry of the intracellular vestibule of the putative transport pathway, which has been proven by electrophysiological experiments to be a gating modulator. Multiple chloride and sodium ions are resolved in the high-resolution structure, identifying novel ion-binding pockets for the first time. Together, our structure takes important steps in elucidating the structural features and regulatory mechanism of SLC26A9, with potential significance in the treatment of cystic fibrosis.

Keywords: Cryoelectron microscopy; Molecular biology.

Fig. 1. Structure of the human SLC26A9 homodimer.

a Cryo-EM map and overall structure of the human SLC26A9 homodimer. Different subunits are colored in pink and blue. b Structure of the human SLC26A9 colored according to the different domains, with the N-terminus (N-ter) in gold, STAS domain in marine, C-terminus (C-ter) in violet, core domain in orange, and gate domain in lime. c Topology of human SLC26A9. The transmembrane (TM) segments are numbered from 1 to 14. The unwound region is formed by the two half-helices TM3 and TM10. An additional helix is located between TM6 and TM5 and is therefore labeled TM5b. The secondary structure of STAS domains is shown. All are colored accordingly.

Fig. 2. C-terminus binding alters transport function.

a The C-terminal sequence is located in the cytosolic vestibule. b Hydrophilic interactions of the C-terminal sequence, which anchored the C-terminal sequence through interaction with TM5 and TM12. c Electrophysiological evidence for C-terminus gating, the cell numbers patched for each group were n = 19, 16, 11, 14, 11, respectively, ***P < 0.001. The sequence of the “peptides” is DLEQEMFGSMFH, which is the same as C-terminal sequence. The sequence of the “control peptide” is YEVHHQKLVFF. The same concentrations of both peptides were applied in the experiments. d Electrophysiology study of mutants that are involved in the C-terminal sequence binding. The currents recorded were –0.79 ± 0.06, –1.84 ± 0.28, –1.37 ± 0.29, –1.49 ± 0.23 pA and the cell numbers patched for each group were n = 19, 6, 8, 6, respectively, *P < 0.05, ***P < 0.001.

Fig. 3. Ions and water molecules are resolved in human SLC26A9 structures.

a Three water molecules are observed in the traditional substrate-binding pocket. Residues participating in coordination are labeled. b A chloride ion is identified near the N-terminus of the TM3 α-helix. c A sodium ion is coordinated through the hydroxyl groups of S107, T127, and Q85. d A chloride ion is bound near the extracellular side of SLC26A9. Residues from the TM1–TM2 loop and TM4 participate in coordination. e Electrophysiology of mutants that are involved in ion coordination. The currents recorded were –0.79 ± 0.06, –0.37 ± 0.03, –0.31 ± 0.02, –0.30 ± 0.02, and –0.26 ± 0.03 pA, and the cell numbers patched for each group were n = 19, 10, 10, 6, and 6, respectively, ****P < 0.0001. f Molecular dynamics simulation analysis of the sodium and chloride ion-binding sites. The TM10, TM1 and TM3, TM8 of the modeled structure are colored in pink and cyan, respectively. Modeled densities of Na⁺ and Cl^– are shown in pink and yellow dots, respectively. The positions of the ions in the cryo-EM structure are illustrated as magenta and green spheres. No apparent disagreement is observed between the modeled and actual positions of the ions. g The distance to site is plotted against time for Cl^– ions. h The distance to site is plotted against time for Na⁺ ions.

Fig. 4. Comparison between the SLC26A9 structure and similar structures.

a A superposition of the structures of human SLC26A9 and mouse Slc26a9. The major difference lies in the α extension of the STAS domain and the C-terminal sequence. b Enlarged view of the α extension. c Enlarged view of the C-terminal sequence. d Human SLC26A9 and SLC26Dg (PDB ID: 5IOF) aligned by the ion permeation pathway. SLC26Dg is colored cyan, and SLC26A9 is colored brown. The substituted residues are colored according to their relative charge, with blue representing more electropositivity and red representing more electronegativity. e Human SLC26A9 and human SLC4A1 (PDB ID: 4YZF) aligned by their gate domains. As SLC4A1 is solved in the outward-facing conformation, conformation differences in the core domains further support the elevator alternative-access model of SLC26A9. TM3 and TM10 are enlarged to depict the movement.