The Role of Conserved GRD Motif in the Allosteric Regulation of Ycf1 Transport Activity and Protein Function

Abstract

Multidrug resistance is a rising issue in the healthcare field and presents itself in bacteria, fungi, and mammalian cells. Clinical isolates of multidrug resistant cells reveal an upregulation in ATP-binding cassette (ABC) proteins, a superfamily of membrane proteins that use ATP to actively transport substrate across biological membranes. Notably, the ABC-C subfamily of ABC transporters are heavily associated with multidrug resistance, including multidrug resistance-associated protein (MRP1), which is heavily involved in anticancer drug resistance in humans. The transport cycle of ABC exporters has been extensively studied and includes the following basic steps: substrate binding, ATP binding, ATP hydrolysis, and substrate efflux. It is unknown how substrate binding traffics ATP to the transporter, however, a conserved sequence on the intracellular loops (ICLs) of the exporter, referred to as the GRD motif, could allosterically regulate ABC transporter function. The role of this motif in a bacterial TAP1/2 homolog, TmrAB, was found to directly impact ATPase activity and substrate transport, and a mechanism for how this motif affects protein function was predicted. A functional and structural ortholog of MRP1, yeast cadmium factor gene 1 (YCF1), contains this conserved sequence, however, the role of this motif in the ABC-C subfamily has not been determined. My studies show that the GRD motif located on ICL1 in Ycf1 is required for ATPase activity in the absence and presence of substrate.