is consistent with the role of hydrophobic side-chains in determining the size of the alcohol-binding pocket. Second, the IRK1-MPD structure indicates that hydrogen-bonds form between MPD and the backbone carbonyl of P244, Y242 via a water and a hydroxyl of Y337 (Fig. 1)21. At the homologous position for IRK1-Y242, GIRK2 contains a Phe (F254), which indicates that this hydrogen-bond triangle may not be essential. Additionally, we found that MPD-mediated activation was not affected by Y349W mutation at the homologous position of IRK1-Y337) (Supplemental Fig. S5). Therefore, it is possible that the carbonyl group of Pro in the βD-βE ribbon is the linchpin that stabilizes alcohol in the pocket via hydrogen-bonding. Unnatural amino acid mutagenesis38 would be needed to further establish the importance of this hydrogen-bond interaction in stabilizing alcohol.
