16 research outputs found
A single active catalytic site is sufficient to promote transport in P-glycoprotein
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
Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter
ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide binding domains (NBDs)(1,2). ABC exporters are present in both prokaryotes and eukaryotes with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases(3,4). TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus homologous to various multidrug transporters and containing one degenerate site with a non-catalytic residue next to the Walker B motif(5). Here we report a subnanometer resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single particle electron cryomicroscopy (cryo-EM). The reconstructions clearly resolved characteristic features of ABC transporters, including helices in the transmembrane domain (TMD) and NBDs. A cavity in the TMD is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two NBDs remain in contact via their C-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two NBDs during the transition from the inward facing to outward facing conformations