National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R00GM114245
United States
Citation
Journal: Nat Commun / Year: 2022 Title: The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation. Authors: Nitesh Kumar Khandelwal / Cinthia R Millan / Samantha I Zangari / Samantha Avila / Dewight Williams / Tarjani M Thaker / Thomas M Tomasiak / Abstract: Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of ...Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.
Name: Metal resistance protein YCF1 / type: protein_or_peptide / ID: 1 Details: Phosphorylated residues (S908, T911 and S914) are present in the structure file. Number of copies: 1 / Enantiomer: LEVO / EC number: ABC-type Cd2+ transporter
Details: Solution were made fresh in cold distilled water and final pH was adjusted to 7.0 with HCl of cold buffer. The digitonin detergent was added to final .06 % in buffer after pH adjustment.
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 283.15 K / Instrument: LEICA EM GP
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Electron microscopy
Microscope
TFS KRIOS
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8499 / Average exposure time: 2.9 sec. / Average electron dose: 54.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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