Abstract 898: Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1).

Abstract

Abstract P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) efflux transporter implicated in the development of multidrug resistance in cancer cells. The catalytic cycle of P-gp is still not fully understood. Both the C431 and C1074 residues in the Walker A motifs of nucleotide-binding domains (NBDs) are highly conserved in P-gp from different species and these residues are excellent reporter sites to study the interaction between the two NBDs during the catalytic cycle. Using homo-bifunctional crosslinkers, disulfide crosslinking of the 431C and 1074C residues in a cysless background can be observed in the presence of copper phenanthroline (CuP) and M17M, which have spacer arm lengths of 0 Å and 25 Å, respectively. However, crosslinking with M17M was prevented in the ADP-vanadate-trapped (closed) conformation, indicating that the distance between 431C and 1074C or the accessibility of these residues changes upon ATP hydrolysis. In addition, the accessibility of the residues, separately or together (431C/1074C) in both the apo and closed conformations was determined with fluorescein-5-maleimide (FM) and methanethio-sulfonate derivatives of rhodamine (MTS-rhodamine) and verapamil (MTS-verapamil), which exhibit different physical properties. The crosslinking of cysteine residues with thiol-reactive compounds is observed both in apo (open) and ADP-vanadate-trapped (closed) conformations, though at a reduced level in the latter case, indicating the flexible nature of the cysteines in Walker A domains. Consistent with these findings, the docking of FM in a homology model of human P-gp ATP sites shows that the sulfur atoms of 431C or 1074C can be oriented either facing the ATP pocket or facing away from the pocket. A different degree of labeling was observed between the two residues; for 1074C having 1.4- fold and 2-fold higher labeling with FM compared to 431C in the apo and closed conformations respectively, suggesting that accessibility of 1074C is greater than that of 431C in both conformations. In the presence of the transport substrate cyclosporine A (50 μM), crosslinking is still observed with CuP and M17M, suggesting that the binding of substrates to the drug-binding site 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 human P-gp are flexible and differentially accessible in the apo and closed conformations. In addition, the NBDs of human P-gp are closer together (20-25 Å) than the NBDs observed in the X-ray structure of mouse P-gp (36 Å). Citation Format: Hong-May Sim, Jaya Bhatnagar, Samantha A. Green, Adam Gonzalez, Eduardo E. Chufan, Suresh V. Ambudkar. Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 898. doi:10.1158/1538-7445.AM2013-898