Figure 7. CALHM6 structure.

(A) Ribbon representation of the decameric CALHM6 structure viewed from within the membrane. Subunits are colored in red and light blue. The C-terminus following CTH is colored in green. Inset (below) shows a close-up of this region with cryo-EM density superimposed. (B) View of the CALHM structure from the extracellular side. (C) Slice through the pore of the CALHM6 structure. B, C, TM1, which has moved towards the pore axis is labeled. A, C, the membrane boundary is indicated. (D) Superposition of single subunits of the CALHM4 and CALHM6 structures illustrating conformational changes. Coloring is as in Figure 5A with CALHM4 shown in brighter shades of the same color. Secondary structure elements are labeled and the hinge for the movement of TM1 is indicated by an asterisk. (E) Sequence alignment of the end of TM1 and the following loop of CALHM paralogs. Selected conserved residues are colored in green and red. Numbering corresponds to residues indicated in panels F and G. Asterisk marks the hinge region displayed in D. Close-up of the extracellular region involved in conformational changes in (F), the cylindrical conformation displayed in CALHM4 and (G), the conical conformation displayed in CALHM6. Residues contributing to a cluster of aromatic residues on TM1-3 and a conserved disulfide bridge are shown as sticks. Selected positions highlighted in E are labeled. F, G, Coloring is as in Figure 5A. (H) Slice through the pore region of the CALHM6 decamer viewed from within the membrane. Shown is non-averaged density at low contour to highlight the location of diffuse density within the pore. A plot of the density along the pore axis of CALHM6 is shown in red, the corresponding density in CALHM4 is shown as a dashed blue line for comparison. The two maxima in the CALHM6 density are shifted towards the intracellular side. The density corresponding to the headgroups of the outer leaflet of the bilayer in CALHM4 is absent. Density at the location of the headgroup region at the inner leaflet of the bilayer and further towards the intracellular side could correspond to either lipids or to the poorly ordered N-terminus.

Figure 7—figure supplement 1. Features of the CALHM6 structure.

(A) Stereo view of a superposition of the CALHM4 and CALHM6 decamers. The proteins are shown as Cα-traces. Coloring is as in Figures 4 and 6. (B) Close-up of the conformational change in TM3 from the cylindrical conformation displayed in CALHM4 to the conical conformation displayed in CALHM6. Right panel shows ribbon, left panel a stick model of TM3 with side chains truncated to their Cβ position except for a conserved proline at the site of conformational changes where the entire side chain is displayed. (C) Close-up of the conformational change of TM1 with molecules displayed as ribbons (left) and interaction region of TM1 and TM3 in the CALHM4 structure with the protein shown as Cα trace and side chains of interacting residues as sticks (right). B and C, coloring of ribbons is as in Figure 7D with secondary structure elements in CALHM4 shown in brighter shades of the same color. (D) Sequence alignments of TM1 and TM3 of different CALHM paralogs. The conserved proline displayed in B is colored in red, selected residues at the TM1 TM3 interaction interface displayed in C are colored in green. B, C, Numbering corresponds to positions labeled in D. (E) Hypothetical pore shapes of the conical conformation displayed in the CALHM6 structure. The disordered N-terminal helix NH (green) was modeled in different conformations. Left, ‘kinked’ conformation where the mutual relationship between TM1 and NH is as in CALHM4, center left, ‘extended’ conformation where NH has changed its conformation to extend the TM1 helix. Center right, ‘clogged’ conformation where the mobile NH helix has moved towards the pore axis. The 10-fold symmetry is maintained in the ‘kinked’ and ‘extended’ conformations and broken for NH in the ‘clogged’ conformation to improve protein interactions. Opposite subunits of the decameric structures are displayed. Right, pore dimensions of the different modeled conformations of CALHM6 calculated with HOLE (Smart et al., 1996) compared to the CALHM4 decamer (red dashed) display similar constricting pore radius as for CALHM4 for ‘kinked’ and ‘extended’ conformations and an occluded pore in case of the ‘clogged’ conformation.