Application of Cysteine Scanning Mutagenesis to the Multidrug Resistance Protein (MRP)1

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Theis, Ashley
Multidrug Resistance , ABC Transporter , Cancer , Cysteine
Multidrug resistance protein (MRP)1, a member of the ABCC branch of the ATP-binding cassette (ABC) superfamily of transporters, can confer resistance to a broad spectrum of chemotherapeutic agents. In addition to the core functional unit of ABC transporters that consists of two membrane spanning domains (MSD) and two nucleotide binding domains (NBD), MRP1 contains a third MSD (MSD0) resulting in the following arrangement: NH2-MSD-(MSD-NBD)2. In lieu of high-resolution structural information for MRP1, cysteine scanning mutagenesis (CSM) was applied to MRP1 and involves the development of a functional template devoid of cysteines into which paired cysteines can be introduced. Previous attempts to create a functional, cys-less template of MRP1 demonstrated that cysteines in MSD0 were structurally and functional important (1;2). However, given that MRP1 lacking MSD0 remains functional, a partially functional, cys-less MRP1 lacking this domain has been expressed in yeast (3-5). Given these results, with the ultimate goal of applying CSM to MRP1 in its entirety, we investigated the endogenous cysteines within MSD0 and co-expressed MRP1 half-molecules and validated these potential CSM templates by transport and ATP binding/hydrolysis assays. Mutation of cysteines within the core of MRP1 had detrimental effects on MRP1 transport activity and further mutation of cysteines by domain revealed that wild-type activity was retained in an MSD0-less MRP1 dual lacking cysteines in both NBDs. This construct was used for introduction of cysteines on juxtaposed faces of the NBD1:NBD2 heterodimer at positions 775 and 1329; comparable residues in the related Cystic Fibrosis Transmembrane Regulator (CFTR/ABCC7) have been suggested to be evolutionarily coupled and joined by a hydrogen bond, maintained in structures of related proteins (6). Unfortunately, functional assays revealed that introduction of cysteines at these positions greatly reduced transport activity of MRP1 and diminished trapping of nucleotide at both NBDs. Finally, alanine substitution of the seven cysteines in MSD0 was not without effect and cellular trafficking assays, co-expression studies and SDS-PAGE analysis suggested an altered conformation of this domain. In addition, a disulfide pair of Cys7 and Cys32 was suggested by these experiments in MSD0 and further supported by examination of these mutants in full-length MRP1.
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