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

• 成果类型: Article
• 署名作者: Quail, Sarah E. S.; Bickers, Sarah C.; Tymczak, Agatha; Eid, Maya Michelle; Kanelis, Voula
• 署名单位: University of Toronto; University of Toronto; University Toronto Mississauga; University of Toronto
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2532795123
• 发表日期: 2026-09-01
• 页码: e2532795123
• 关键词: ABC transporter; intrinsically disordered protein; phosphorylation; NMR spectroscopy; nucleotide binding domain; saccharomyces-cerevisiae; ABC TRANSPORTERS; protein; cftr; detoxification; DYNAMICS; disorder; glutathione; mutations; complexes

摘要

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.