Abstract
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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Author Contributions: R.S.A.C and T.M.T designed research; R.S.A.C conducted protein expression and purification, Cryo-EM studies, viability assay, ATPase activity assay, mass spectrometry, data analysis and manuscript preparation. M.S.I.R performed protein purification viability assay and ATPase activity assay. N.K.K benchmarked protein purification and supported cryo-EM studies. R.S.A.C, M.S.I.R and T.M.T prepared the manuscript.
Competing Interest Statement: The authors declare no competing interest.
Mistakes on the reference numbering. Add techincal information in the material and methods section. To add relevant complementary references in the boyd of the text as well as on the material and methods.