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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
journal contribution
posted on 2013-10-15, 00:00 authored by Hong-May Sim, Jaya Bhatnagar, Eduardo E. Chufan, Khyati Kapoor, Suresh V. AmbudkarP-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.