Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42

Fig. 1. Cryo-EM structure of a full-length human ClC-2 homodimer. a Front view and top view of ClC-2 cryo-EM density map in homodimer form. Two protomers are shown in brown and purple, respectively. b Front view and top view of the ClC-2 atomic model. Color scheme is the same as in (a). The obtained ClC-2 structure can be divided into transmembrane domain (TMD) and the cytosolic C-terminal domain (CTD). c Overall topology of human ClC-2 with transmembrane helices (labeled as A-R) and two cytosolic cystathionine beta synthase (CBS) domains. d Three-dimensional arrangement of transmembrane helices of one protomer as shown in (c).

Fig. 2. Transmembrane domain of the ClC-2 channels in the apo state. a Overall structure of the transmembrane domain of ClC-2 homodimer. Protomers are colored in brown and purple, respectively. The chloride channel was identified by CAVER plugin52 in PyMOL (https://pymol.org/2/) and shown as cyan dots. Only one chloride channel was shown for simplicity. b Enlarged map of the chloride channel, where cyan balls represent three conserved putative chloride ion binding sites inferred from sequences (from top to bottom: Sext, Scen, and Sint). The regions of the selectivity filter (Y553 and S162) and the fast gate (E205) are labeled. c The pore radius along the chloride pathway from the extracellular side to the cytosol is visualized in the chart. The selectivity filter formed by S162, Y553 and E205 is highlighted. E205 was mutated to alanine during the pore radius calculation to reserve the pore connectivity. Source data are provided as a Source Data file. d–f Comparison of the channel gating of human ClC-2 with (d) human ClC-1 (PDB ID 6COY)18, (e) bovine ClC-K (PDB ID 5TQQ)16, and (f) human ClC-7 (PDB ID 7JM7)21. ClC-2 was colored in purple. Human ClC-1, bovine ClC-K and human ClC-7 were…

Fig. 3. Transmembrane domain of the AK-42 inhibitor-bound ClC-2. a Front view and top view of the AK-42 bound ClC-2 transmembrane domain. Two protomers are shown in brown and purple, respectively. AK-42 is shown in green sphere. b Close-up view of the chloride channel with AK-42 bound. The AK-42 inhibitor is located above the Sext site and blocks the Cl− channel. Residues S162, E205, L552, Y553 are shown in stick. AK-42 are shown in green stick. c Conformational change of M460 induced by AK-42. The black dashed line indicates the side chain of M460 in the apo state, which moves upward upon AK-42 bound. Densities of surrounding residues are shown in gray. d Interactions between ClC-2 and AK-42 were analyzed by the LigPlot+ program56. K394 and AK-42 form a hydrogen bond with a distance of 2.57 Å. e ClC-2 residues around the AK-42 inhibitor and homologous residues in other human ClC proteins were extracted and compared. * indicates the three most critical residues in the binding with AK-42 identified in per-residue energy decomposition shown in Supplementary Fig. 7i. f Mapping of identified key residues to the cryo-EM model. Residues identified both in the previous docked models and in the…

Fig. 4. Mapping of disease-causing mutations in the ClC-2 structure. a Labeling of genetic mutation sites (purple color balls) on one protomer of the ClC-2 dimer. The chloride channel and AK-42 inhibitor were also shown. b Close-up view of disease-related mutation sites in the transmembrane domain of ClC-2. Those sites include G98, G199, G466, R471, A500 and G503. c Close-up view of disease-related mutation sites in the para-membrane regions and CBS domains. Those sites include R68, R73, F82, R172, V174, R235, R577, H590, R644, R646 and R653. d, e Proposed model to explain the direct blocking mechanism of AK-42 in ClC-2. Upon binding of AK-42 to ClC-2, the side chain of M460 residue adopts another conformation, however the overall structure of ClC-2 does not change significantly. Residues E205, Y553, M460 and two putative chloride binding sites are shown.