Figure 4. Glutamate gate (Glu_gate) and Cl⁻-binding sites of CLC-1.

(A) View (stereo) into the selectivity filter of CLC-1. αN, αF, αR, and αC-D segments (Cα trace and side chains) are shown in cyan, salmon, yellow, and olive, respectively. The side chains of Glu_gate, Tyr_C, and Ser_C are represented with balls and sticks. Cl⁻-binding sites are indicated by green (S_ext and S_int) and gray (S_cen) spheres. The cryo-EM density map is shown in mesh (S_ext and S_int in magenta and the rest in gray). (B) Water-accessible regions in the filter region, probed by Hollow (Ho and Gruswitz, 2008), are shown with purple (extracellular vestibule) and blue (intracellular vestibule) dots. Glu_gate is represented in ball-and-stick. (C) Comparison of Glu_gate positions between the CLC-1 channel and CLC transporters. The amino acid segments 146–149 and 355–358 forming the anion selectivity filter were aligned between structures. Cα-traces of the segments are shown with the Glu_gate side chains in ball-and-stick representation. Blue, CLC-1. Light orange, WT EcCLC (PDB ID: 1OTS). Yellow, EcCLC E148Q mutant (PDB ID: 1OTU). Magenta, CmCLC (PDB ID: 3ORG). Gray spheres represent the positions of Cl⁻ ions seen in EcCLC E148Q mutant (S_ext and S_cen). Note that the Cl⁻ ion at CLC-1’s S_ext (not shown) essentially coincides with S_ext of EcCLC.

Figure 4—figure supplement 1. Comparison of the anion selectivity filter and Glu_gate between CLC proteins.

(A–D) As in Figure 3B, but focused on Glu_gate and neighboring hydrophobic amino acids (stereo views with the same view angle). (B–D) also include superimposed Glu_gate of CLC-1 in pink semi-transparent sticks, to show steric clashes with neighboring hydrophobic amino acid side chains of other CLC proteins. The green spheres indicate the position of S_ext or S_cen. Note that in the EcCLC and CmCLC structures, Glu_gate’s side chain occupies S_ext and S_cen, respectively, and in the bCLC-K, S_ext is unoccupied.