*2.5. Sidechain Properties in the Allosteric Corkscrew*

In a recent review [24], it was noted that composition of residues in the polar relay of ABCG2 and of ABCG5/G8 differs, with ABCG8 having a relatively high number of charged residues and ABCG2 a relatively low number. In residues with the conservation pattern (ABCG1, ABCG4), (ABCG2), (ABCG5), (ABCG8) this pattern is reiterated, ABCG8 having seven charged residues, ABCG5 five and ABCG2 one (Figure 5b). Perhaps notably, the only charged residue in ABCG2 with this conservation pattern is R482, mutation of which is long associated with altered substrate specificity [11,22,23].

An even greater discriminant between ABCG2 and other ABCG members is that 15 of the 33 residues with this conservation pattern are polar, hydroxylated residues (serine or threonine) in ABCG2 (Supplementary Figure S3). There are relatively few hydroxylated amino acids in these positions for other ABCGs (ABCG1/4: 5, ABCG5: 5, ABCG8: 3). Do these dissimilarities in the corkscrew of type II divergent residues contribute to differences in protein function? Polar and ionisable residues can drive specific helix oligomerisation, but this does not include serine or threonine alone [25–27] and we did not observe the specific motifs predicted to drive helix association [28,29]. Rather, an intriguing possibility is that serine and threonine form intra-helical hydrogen bonds, which can bend the helix in

certain conformations, lending ABCG2 unusual flexibility in this region. Other residues significantly contributing to flexibility, such as glycine and proline, are no more common in other proteins compared with ABCG2. This extra flexibility could permit the binding and transport of diverse sizes of substrates, coupled to allosteric motions communicated through this network. Similar influence of hydroxylated amino acids in driving substratespecific conformational changes is observed in some GPCRs [30–33].
