Mapping conformational changes associated with the catalytic cycle of human P-glycoprotein (ABCB1)

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NCI	CANCER-2

Authors

• J Bhatnagar
• H Sim
• K Kapoor
• E Chufan
• S Ohnuma
• E Georgieva
• P Borbat
• J Freed
• Z Sauna
• S Ambudkar

Abstract

P-glycoprotein (Pgp, ABCB1) is an ABC transporter that uses the energy of ATP hydrolysis to efflux a variety of amphipathic substrates including anti-cancer agents from the cell. In this study, we have used disulfide crosslinking and Pulsed Dipolar Electron Spin Resonance Spectroscopy (PDS) to elucidate the conformational changes associated with the catalytic cycle. We generated a library of twenty five double cysteine mutants in a cys-less background and expressed in baculovirus-insect and mammalian cell expression system. All the mutants were expressed at the cell surface in HeLa cells and were also functionally active. Disulfide crosslinking was performed in crude membranes and each double cysteine mutant of Pgp was tested for its ability to crosslink with ten crosslinkers of varied spacer arm length. The reactivity of cys residues in these mutants was verified by labeling with fluorescein maleimide and these mutants were also studied with PDS to obtain long-range distance information (20-65Å) between a pair of spin labeled cysteine residues. In five double cysteine mutants (with cys residues in different domains of Pgp) significant modulation of crosslinking and changes in extracellular loops and ATP sites were observed in APO to ADP-vanadate-trapped conformation suggesting high degree of flexibility in Pgp.

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