Studies of the substrate-binding pocket of the human multidrug resistance-associated protein MRP1

Very little information is currently available on the structure/function of MRP1. The structure of the MRP1 substrate-binding pocket has not been elucidated, and the properties of this pocket are poorly understood. The long-term aim of this work is to identify the substrate-binding pocket of MRP1 and determine its structure. Here, I started the identification of MRP1 transmembrane helices involved in substrate binding using a combination of molecular biological and biochemical techniques that include Cys-scanning mutagenesis, Cys modification with thiol reagents, replacement of MRP1 alpha helices with a poly-Ala sequence, and assessment of residue accessibility to solvent bath solution.; First, I developed a Saccharomyces cerevisiae MRP1 expression system that allows for the rapid generation and screening of mutants, as well as simple purification of MRP1. Second, I developed a functional Cys-less variant of MRP1. The functional properties of this Cys-less MRP1 are similar to those of the wild-type MRP1. Third, I tentatively identified transmembrane segment 17 as a helix that forms part of the MRP1 substrate-binding pocket, whereas transmembrane segment 16 does not. Forth, I developed a new poly-Ala helix-scanning technique characterized by the replacement of complete transmembrane segments with poly-Ala alpha helices. Data using this technique indicate that as long as transmembrane segment 17 is an alpha helix, without sequence specificity, the interaction of MRP1 with its high-affinity substrate leukotriene C4 remains intact. Residues in transmembrane segment 17, though, seem to be involved in the interaction of MRP1 with an estradiol glucuronide conjugate substrate. Fifth, experiments using Cys-scanning mutagenesis coupled to the use of thiol-selective crosslinking reagents suggest that the multidrug binding pocket of MRP1 is large enough to accommodate two substrates simultaneously. These and future results will help providing a better understanding of the structure of MRP1 and will enable us to design better anticancer drugs to improve the efficiency of anticancer therapy in multidrug-resistant tumors. (Abstract shortened by UMI.)...