MULTIDRUG RESISTANCE PROTEIN: P-GLYCOPROTEIN - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 227

Modulators are as diverse structurally as substrates. 27 They appear to interact with the
same binding site(s) as drugs and compete with them for transport. Many modulators
(e.g., verapamil, cyclosporin A, trans -flupenthixol) are themselves transported by the
protein. Cells are generally not resistant to killing by modulators, but they are killed
by MDR drugs in combination with modulators. The way in which modulators exert
their action at the molecular level is still not well understood.
10.5. P-GLYCOPROTEIN STRUCTURE
Like many other ABC proteins, 28 , 29 Pgp comprises two membrane-bound domains,
each made up of six transmembrane (TM) helices and two cytoplasmic nucleotide-
binding (NB) domains which bind and hydrolyze ATP (Figure 10.1 a ). The topology
of Pgp was established using molecular biological methods such as Cys mutations and
insertion of glycosylation sites. 30 , 31 Earlier studies using various heterologous expres-
sion systems suggested alternative topologies in which putative TM segments were
displaced outside the membrane, however, it seems likely that these arrangements
were the result of misfolding and do not reflect the true topology of the transporter in
vivo. 32 The TM regions from both halves of Pgp form the drug-binding region of the
protein, 33 and drugs enter this binding pocket from the lipid bilayer. 34
High-resolution x-ray crystal structures of two ABC proteins, the catalytic domains
of the DNA repair enzyme Rad50cd 35 and the vitamin B12 importer BtuCD, 36 showed
that the two NB domains were in close contact to form a dimeric structure. Two
molecules of ATP were bound at the dimer interface, with each binding site comprising
the Walker A and B motifs of the cis -NB domain and the LSGGQ signature C motif
of the trans -NB domain. This “sandwich dimer” structure has also been observed
for the isolated NB domain of the ABC protein MJ0796, which forms a stable dimer
when the ATPase activity of the protein is inactivated by the mutation E171Q. 37 It
seems likely that this dimerization process plays a critical role in the catalytic cycle
of the ABC proteins and may be closely tied to the power stroke. 29
No high resolution x-ray crystal structure is available for Pgp. Early work by
Rosenberg et al. using electron microscopy (EM) single-particle image analysis of
purified Pgp produced a very low resolution structure which suggested the existence
of a large 5-nm-diameter central pore in the protein. 38 This pore was closed at the
cytoplasmic face of the membrane, forming an aqueous chamber within the membrane
from which entry points to the membrane lipid were observed. Two widely separated
3-nm lobes on the cytoplasmic side of the membrane were thought to represent the NB
domains. This structure was at odds with both biochemical studies, which suggested
kinetic cooperativity between the two catalytic sites, and the high-resolution x-ray
crystal structures of other ABC proteins described above, which showed close physical
association of the two NB domains. Fluorescence resonance energy transfer (FRET)
studies in which two different fluorescent probes were covalently linked to each
Walker A motif Cys residue also indicated that the positioning of the two NB domains
is compatible with the sandwich dimer model (Figure 10.1 b ), 39 and Urbatsch et al.
found that the two Walker A Cys residues could readily cross-link spontaneously. 40