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Sci Rep. 2016 Apr 27;6:24810. doi: 10.1038/srep24810.

A single active catalytic site is sufficient to promote transport in P-glycoprotein.

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Department of Biophysics and Cell Biology, University of Debrecen, P.O. Box 400, Debrecen H-4002, Hungary.
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Hungary.
Institute of Horticulture, University of Debrecen, P.O. Box 36, H-4015 Debrecen, Hungary.
Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, H-1525 Budapest, Hungary.
MTA-DE Momentum, Genome Architecture and Recombination Research Group, Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen H-4032, Hungary.
MTA-SE Ion Channel Research Group, Budapest H-1094, Hungary.
Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.


P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.

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