Conserved Walker A Cysteines 431 and 1074 in Human P-Glycoprotein Are Accessible to Thiol-Specific Agents in the Apo and ADP-Vanadate Trapped Conformations

Abstract

P-Glycoprotein (P-gp) is an ATP-binding cassette efflux transporter involved in the development of multidrug resistance in cancer cells. Although the mechanism of P-gp efflux has been extensively studied, aspects of its catalytic and transport cycle are still unclear. In this study, we used conserved C431 and C1074 in the Walker A motif of nucleotide-binding domains (NBDs) as reporter sites to interrogate the interaction between the two NBDs during the catalytic cycle. Disulfide cross-linking of the C431 and C1074 residues in a Cys-less background can be observed in the presence of M14M and M17M cross-linkers, which have spacer arm lengths of 20 and 25 Å, respectively. However, cross-linking with both cross-linkers was prevented in the ADP-vanadate trapped (closed) conformation. Both C431 and C1074 alone or together (double mutant) in the apo and closed conformations were found to be accessible to fluorescein 5-maleimide (FM) and methanethiosulfonate derivatives of rhodamine and verapamil. In addition, C1074 showed 1.4- and 2-fold higher degrees of FM labeling than C431 in the apo and closed conformations, respectively, demonstrating that C1074 is more accessible than C431 in both conformations. In the presence of P-gp substrates, cross-linking with M17M is still observed, suggesting that binding of substrate in the transmembrane domains does not change the accessibility of the cysteines in the NBDs. In summary, the cysteines in the Walker A motifs of NBDs of human P-gp are differentially accessible to thiol-specific agents in the apo and closed conformations.