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bioRxiv · 10.1101/2021.06.18.449046

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

Khandelwal, N. K.·Millan, C. R.·Zangari, S. I.·Avila, S.·Williams, D.·Thake, T. M. r.·Tomasiak, T. M.
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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[A] and 4.0[A] in distinct inward-facing open conformations capturing a previously unobserved ordered state 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 domain. 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 long-sought after insights into how R-domains control ABCC transporter activity.

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Khandelwal, N. K., Millan, C. R., Zangari, S. I., Avila, S., Williams, D., Thake, T. M. r., Tomasiak, T. M.. 2021-06-18. The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation. https://doi.org/10.1101/2021.06.18.449046

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