Structural Studies of Multidrug Resistance Protein 1 Using “Almost” Cysless Template

Abstract

Multidrug resistance protein, MRP1 (ABCC1) is a broad-spectrum ATP-binding cassette transporter that plays a major role in defense against dietary and environmental toxicants, in addition to contributing toward multidrug resistance of certain types of malignancy. Elucidating the molecular structure of hMRP1 is key to determining its mechanism of substrate recognition and transport. Here, we report the first successful attempt using cysteine-scanning mutagenesis coupled with cross-linking studies to probe the structure of hMRP1 in its native environment of the cell membrane or in membrane vesicles. We have established that an active 3Cys ΔMRP1 (MRP204-1531) mutant, described in previous studies from our laboratory, is a suitable template with which to generate single- and double-cysteine mutants for performing cysteine mutagenesis studies. We have now used 3Cys ΔMRP1 to probe the arrangement of several TM segments, as well as the location of individual amino acids in these regions. Cysteine residues were introduced into TMs 8, 14, 15, and 16 of 3Cys ΔMRP1. 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); and R433C (TM8) and T1082C (TM14). The aqueous accessibility of these residues and the possible implications of the differences between the open and closed states of the protein are also discussed. Moreover, using competition experiments involving a well characterized substrate and a cross-linking reagent for probing the Cys388/ I1193C mutant, we have defined these amino acid positions as a component of the potential site for estrone sulfate binding.