TY - JOUR T1 - Structural studies of Multidrug Resistance Protein 1 (MRP1) using "almost" cysless template JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.117.078709 SP - dmd.117.078709 AU - Daria N. Trofimova AU - Roger G. Deeley Y1 - 2018/01/01 UR - http://dmd.aspetjournals.org/content/early/2018/03/23/dmd.117.078709.abstract N2 - Multidrug resistance protein, MRP1 (ABCC1), is a broad spectrum ATP-Binding Cassette (ABC) transporter that plays a major role in defence against dietary and environmental toxicants, in addition to contributing towards multidrug resistance of certain types of malignancy. There is no high-resolution crystal structure of human MRP1 but a model has been generated by computational comparative modeling based on the crystal structure of the bacterial transporter S. aureus Sav1866. The model has been used extensively in interpreting the functional results of mutational studies. However, no direct biochemical data exist at present to support the model’s validity. Here, we report the first successful attempt using cysteine-scanning mutagenesis coupled with cross-linking studies to probe the structure of MRP1. An active 3Cys ΔMRP1 mutant was used as a template to generate single and double cysteine mutants that contained cysteine residues introduced into TMs 8, 14, 15, and 16. The mutants were then subjected to chemical cross-linking analyses, and cross-linking was detected between the following cysteine pairs: Cys388 (TM7) and I1193C (TM16); Cys388 (TM7) and E1144C (TM15); R433C (TM8) and E1144C (TM15); R433C (TM8) and T1082C (TM14). Our results regarding the packing of TM helices at the membrane/cytosol interface are consistent with the predicted packing of TMs in the Sav1866-based model of MRP1. Implications of the aqueous accessibility of these residues and possible differences between the open and closed states of the protein are also discussed. ER -