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

Abstract

Many ATP binding cassette (ABC) proteins function in active transport of solutes across biological membranes. At minimum, ABC proteins are composed of two repeats, each consisting 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 electron cryo-microscopy maps, and thus information about how R region phosphorylation controls ABC transporter activity is missing. Here we employ nuclear magnetic resonance (NMR) spectroscopy to discern the structural features and interactions of the R region from the yeast cadmium factor 1 protein (Ycf1p), a member of the C subfamily of ABC proteins 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, for the first time, NBD2. NBD/R region interactions depend on the phosphorylation state of the R region and the nucleotide-bound and oligomeric states of the NBDs, indicating how R region interactions change in the transport cycle. Complementary biochemical studies show that R region phosphorylation affects the ATPase activity of the NBDs. The molecular-level information provided by the solution NMR studies presented allow for a more complete model of how R region phosphorylation modulates the activities of Ycf1p and related ABC proteins.