Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Abstract

Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

Fig. 1. Structure determination of dephosphorylated Ycf1.

A Size exclusion chromatography profile of dephosphorylated Ycf1. This trace is representative of two replicates. Source data are provided as a Source Data file. B Cryo-EM density of the dephosphorylated Ycf1 structure. TMD0 is colored yellow, TMD1 and NBD1 are colored white, TMD2 and NBD2 are colored wheat, the lasso domains are colored green, and the regulatory domain (R-domain) is purple. C Topology cartoon illustrating the domain architecture of Ycf1 with residue numbers at domain boundaries indicated. D Cartoon of the dephosphorylated Ycf1 structure.

Fig. 2. Structure comparison of Ycf1 in dephosphorylated and phosphorylated states.

A Cryo-EM density and (B) model of dephosphorylated and phosphorylated Ycf1 structures IFwide (PDBID: 7M69) and IFnarrow (PDBID:7M68) colored as in Fig. 1. The area of missing density for the R-domain in dephosphorylated state around NBD is highlighted with a hashed box.

Fig. 3. Representation of the R-domain in the Ycf1 substrate cavity.

A A space-filling model of Ycf1 with a transverse section of the TMD removed to show the R-domain from a side view (left) and bottom view of the NBDs (right). B Electrostatic interactions between the R-domain and the substrate binding cavity. The R-domain backbone is colored purple, with interacting residues and the Ycf1 backbone colored white. Amino acid side chains interacting between the R-domain and Ycf1 are colored based on physical amino acid properties (Hydrophobic residues are colored orange, positively charged amino residues are in cyan, negatively charged residues are in pink, and polar-uncharged residues colored in green. C Hydrophobic interactions with the same color scheme as in (B). Detailed view of R-domain hydrophobic interactions with the TMD region of Ycf1 and. D the C-terminal region of the R-domain.

Fig. 4. Comparison of transport substrate binding pocket in dephosphorylated Ycf1, phosphorylated Ycf1, and substrate bound MRP1.

A Solvent accessible space-filling model of the Ycf1 substrate cavity. B Ligplot detailing specific interactions between dephosphorylated Ycf1 and the R-domain in its binding cavity. C Comparisons of the phosphorylated Ycf1 R-domain binding site to MRP1 (PDB IB: 5UJA). D Detailed view of the dephosphorylated Ycf1 R-domain interaction in the substrate binding pocket. E Interaction network of the glutathione moiety in Leukotriene C4.

Fig. 5. Functional characterization of Ycf1 activity upon dephosphorylation and mutation.

A ATPase rates in different Ycf1 phosphorylation states and Ycf1 mutants. ATPase rates of Ycf1 with and without glutathione acceleration. All values are the mean of four replicates ± standard deviation (S.D.) Source data are provided as a Source Data file. B Spot assays showing survival of cells with Ycf1 mutants. First spot of each mutant is 0.1 OD at 600 followed by 5-fold serial dilution as represented by black triangle. C Limited proteolysis of phosphorylated Ycf1, dephosphorylated Ycf1, and R-domain mutants.

Fig. 6. Proposed model for R-domain autoinhibition and activation.

A Overall cycle of Ycf1 transitioning from a dephosphorylated state to a phosphorylated state. The R-domain is colored purple, and the substrate cavity is colored blue. B A closeup of the R-domain hairpin loop (purple) is shown with interacting hydrophobic residues (tan).