Site-specific phosphorylation affects the structure and interactions of the Ycf1p R region.

Authors

Sarah E S Quail, Sarah C Bickers, Agatha Tymczak, Maya Michelle Eid, Voula Kanelis

Published in

Proceedings of the National Academy of Sciences of the United States of America. Volume 123. Issue 35. Pages e2532795123. Epub Aug 24, 2026.

Abstract

Many ATP-binding cassette (ABC) proteins function in active transport of solutes across biological membranes. At minimum, ABC proteins contain two repeats of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). In many ABC proteins, the TMD-NBD halves are connected by an intrinsically disordered linker that regulates the activity of the ABC protein through phosphorylation. These regulatory (R) regions are often invisible or at low-resolution in cryo-EM maps. Thus, information about how R region phosphorylation controls ABC transporter activity is missing. Using NMR spectroscopy, we discern the structural features and interactions of the R region from the yeast cadmium factor 1 protein (Ycf1p), a C subfamily ABC protein that is homologous to human multidrug resistance protein 1. Our data show that the entire R region possesses residual secondary structure that changes with phosphorylation, including for often-invisible R region segments. The data demonstrate R region interactions with NBD1 and also with NBD2. NBD/R region interactions depend on the phosphorylation state of the R region and on the nucleotide-bound and oligomeric states of the NBDs, indicating how R region interactions change during the transport cycle. Complementary biochemical studies show that R region phosphorylation affects the ATPase activity of the NBDs. Yeast viability assays highlight the importance of R region residual structure and interactions on Ycf1p activity. The structural, biochemical, and in vivo studies enhance our molecular-level understanding of how R region affects the transport cycle of Ycf1p and related ABC proteins.

PMID:
42636357
Bibliographic data and abstract were imported from PubMed on 25 Aug 2026.

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