Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation

Many ATP-binding cassette (ABC) transporters are regulated by phosphorylation on long and disordered loops which present a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of Yeast Cadmium Factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.2 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and a position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 as well as an interaction with the R-insertion, another regulatory region. We systematically probe these interactions with a linker substitution strategy along the R-domain and find a close match with these interactions and survival under Ycf1-dependent growth conditions. We propose a model where four overlapping phosphorylation sites bridge several regions of Ycf1 to engage in a transport-competent state.