@article{370731c50347440f843069176f2757dd,
title = "The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation",
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 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 {\AA} and 4.0 {\AA} 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.",
author = "Khandelwal, {Nitesh Kumar} and Millan, {Cinthia R.} and Zangari, {Samantha I.} and Samantha Avila and Dewight Williams and Thaker, {Tarjani M.} and Tomasiak, {Thomas M.}",
note = "Funding Information: A portion of this research was supported by NIH grant U24GM129547 and performed at PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work was also supported by grants from the National Institute of General Medicine Sciences (NIH R00 GM11424 awarded to T.M. Tomasiak), the National Institute of Allergy and Infectious Disease (NIH R01 AI156270 awarded to T.M. Tomasiak), NIH S10 OD011981 (Life Sciences North Imaging Facility at the University of Arizona), and NSF1531991 (Eyring Materials Center at Arizona State University). We thank the staff at the Life Sciences North Imaging Facility at the University of Arizona and the Pacific Northwest Center for Cryo-EM (PNCC), especially Theo Humphreys, Nancy Meyer, and Craig Yoshioko at PNCC for assistance with data collection and helpful advice. We thank the Eyring Materials Center at Arizona State University for assistance with cryo-EM data collection of samples. We thank Krishna Parsawar and Cynthia David from the Analytical & Biological Mass Spectrometry Facility at the University of Arizona for their analysis of mass spectrometry-based phosphorylation identification. We also thank Alexei Rohou, Axel Brilot, Tamir Gonen, and Meghna Gupta for helpful discussions on cryo-EM map refinement and model building, and members of the Tomasiak lab and Robert Stroud for helpful discussions and critical reading of this manuscript. Funding Information: A portion of this research was supported by NIH grant U24GM129547 and performed at PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work was also supported by grants from the National Institute of General Medicine Sciences (NIH R00 GM11424?awarded to T.M. Tomasiak), the National Institute of Allergy and Infectious Disease (NIH R01 AI156270?awarded to T.M. Tomasiak), NIH S10 OD011981 (Life Sciences North Imaging Facility at the University of Arizona), and NSF1531991 (Eyring Materials Center at Arizona State University). We thank the staff at the Life Sciences North Imaging Facility at the University of Arizona and the Pacific Northwest Center for Cryo-EM (PNCC), especially Theo Humphreys, Nancy Meyer, and Craig Yoshioko at PNCC for assistance with data collection and helpful advice. We thank the Eyring Materials Center at Arizona State University for assistance with cryo-EM data collection of samples. We thank Krishna Parsawar and Cynthia David?from?the Analytical & Biological Mass Spectrometry Facility at the University of Arizona for their analysis of mass spectrometry-based phosphorylation identification. We also thank Alexei Rohou, Axel Brilot, Tamir Gonen, and Meghna Gupta for helpful discussions on cryo-EM map refinement and model building, and members of the Tomasiak lab and Robert Stroud for helpful discussions and critical reading of this manuscript. Publisher Copyright: {\textcopyright} 2022, The Author(s).",
year = "2022",
month = dec,
doi = "10.1038/s41467-022-28811-w",
language = "English (US)",
volume = "13",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}