*2.6. Conservation of Other Regions*

Electron microscopy and X-ray crystallography data on ABCGs have indicated other structural regions that are proposed to be critical for allosteric communication. We analysed the triple helical bundle between the NBD and TMD, which is considered to be a vital region for transmission of force from ATPase activity to the TMD. This region spans 54 columns in the multiple sequence alignment and 28 different conservation patterns are observed here. The hot-spot helix is most highly conserved, with 40% of its residues being conserved across the alignment, but no other patterns are significantly different here from the alignment as a whole (Supplementary Table S4). Though the triple helical bundle is highly conserved, the whole of it is not conserved across the G subfamily. Nor is it a motif that defines the difference between ABCG members well. Part of this comes from its being less well conserved in ABCG5 and ABCG8 (~70% for each), perhaps indicating that heterodimerisation reduces the evolutionary pressure on some of these positions. This may be particularly true for this region, due to its importance for transmitting force from ATP hydrolysis [34], which is altered in ABCG5/G8 due to the degenerate NBS.

Given the differences between the dimerisation behaviour of ABCG members (i.e., that some form homodimers and others are obligate heterodimers), we inspected the dimerisation interfaces (both at the TMD:TMD and the G-family specific NBD:NBD interface [35] to see if this was reflected in the conservation patterns. Residues within 5 Å of the other protomer in the structures 6VXF (ABCG2) and 5DO7 (ABCG5/G8) were found and the conservation patterns of corresponding columns were examined (Supplementary Table S5). A total of 46 different patterns are represented in this set, with completely conserved again being the most frequently observed. However, none of the patterns makes up a statistically significant fraction, meaning that the dimer interface is not a useful discriminant between ABCG members.

Binding pockets for substrates of ABCG2 and ABCG5/G8 have been identified from their structures. These are compared in Supplementary Figure S4 and Table S6. Interestingly, there is little overlap between the residues contributing to these pockets, with two columns contributing to the binding pockets of both ABCG2 and ABCG5, and two contributing to both ABCG5 and ABCG8. Both of the columns contributing to the pockets of both ABCG2 and ABCG5 have the conservation pattern (ABCG1, ABCG4), (ABCG2), (ABCG5), (ABCG8), so are part of the corkscrew. A further five columns with this conservation pattern contribute to the binding pocket of ABCG2, and another to that of ABCG5.
