Supplementary Figure 6: Sequence conservation of residues in the interface among the TOP domains, the TLC and the S3–S4 extension.

(a) Three views of the PC2 tetramer (looking from top (external face), side (membrane plane) and bottom (cytoplasmic face)). The molecular surface is shown colored by sequence conservation (variable: yellow, highly conserved: dark blue) based on CONSURF analysis of an alignment of 177 PC2 homolog sequences. The external surface displays minimal sequence conservation apart from a small patch on each TLC extension involved in interdomain interaction. In contrast, the cytoplasmic face displays a high degree of conservation around the S6 bundle crossing and within the exposed face of the VSLD four-helix bundle. (b) Detail of the S3b-S4 interface. The VSLD extension is show in ribbon form and colored by sequence conservation. Residues contributing to the conserved interaction site on the TOP β-sheet are labelled. (c) Schematic cartoon ribbon for PC2 monomer colored by conservation viewed in the same orientation as the side view above in (a). The S3b-S4 interaction site with the outer face of the TOP β-sheet is highly conserved. The S3b-S4 interface shown in panel (b) is highlighted. (d) Interaction of the TOP domain with the pore turret region. The pore turret region (ribbons) interacts with the α1 helix and β5 strand of the adjacent TOP domain (molecular surface; regions from the adjacent protomer are indicated by asterisks). Close contacts are made between the S5-PH loop including a direct hydrogen bond between Gln⁶²² and the backbone carbonyl of Phe⁴⁵⁷ located in β5b. Residues in the turret region and the molecular surface from the adjacent TOP domain are colored by sequence conservation. A significant conserved patch maps to the TOP domain region that interacts with the pore turret. (e) Internal face view of PC2 monomer (180° rotation compared to panel (c)) highlighting the conserved nature of the TLC1 hairpin extension (dotted oval). (f) Perpendicular view corresponding to the approximate orientation of a neighbouring monomer in the tetramer. The surface that the TLC1 hairpin (shown in (e)) interacts with on the other end of the neighbouring TOP domain is indicated with dotted oval and labelled ‘2’. (g) Detail of the TLC1 hairpin extension interaction site.