Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Khandelwal, Nitesh Kumar; Tomasiak, Thomas M.

doi:10.1038/s41467-024-46722-w

Title: Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Journal Article · 15 March 2024 · Nature Communications

DOI: https://doi.org/10.1038/s41467-024-46722-w · OSTI ID:2471634

Khandelwal, Nitesh Kumar ^[1];

^[2]

University of Arizona, Tucson, AZ (United States); University of California, San Francisco, CA (United States)
University of Arizona, Tucson, AZ (United States)

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.

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Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 2471634

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 15; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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