MULTIDRUG RESISTANCE PROTEIN: P-GLYCOPROTEIN - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 236

(R123). 112 The two sites interacted with each other allosterically, such that H-site and
R-site drugs mutually stimulated each other's transport, whereas two H-site drugs
inhibited each other's transport, as did two R-site drugs. Later work suggested the
existence of a third allosterically linked drug transport site. 124
Soluble bacterial transcription factors that bind multiple drugs (i.e., QacR, BmrR,
and MarR) have provided intriguing insights into how a single drug-binding site can
accommodate many structurally diverse compounds. 125 Crystallographic studies of
QacR complexes with six drugs showed that the protein contains a large, flexible bind-
ing pocket, rich in aromatic amino acids, but also containing some polar residues. 126
Van der Waals and hydrophobic interactions play a major role in drug-binding, aug-
mented by electrostatic interactions between charged groups on the drug and charged
amino acid side chains. The size and flexibility of the binding pocket allow drugs with
different structures to establish interactions with different subsets of residues. Two
distinct but partially overlapping binding pockets were observed. Later studies showed
that two drugs could bind to the protein simultaneously. 127 Structural studies of the
human xenobiotic nuclear receptor, PXR, showed that the same drug can bind within a
large hydrophobic cavity in three different orientations, each stabilized by a different
complement of polar side chains. 128
Multidrug transport proteins such as Pgp probably bind their substrates using
principles similar to those observed for soluble multidrug-binding proteins. 129 The
crystal structure of the bacterial RND-family multidrug efflux pump AcrB, binding
four structurally diverse drugs, showed that three ligand molecules bind simultane-
ously to a large central cavity, primarily by hydrophobic, aromatic stacking, and van
der Waals interactions. 130 Each drug binds to AcrB using a different subset of amino
acid residues. Studies using Cys mutants and thiol-reactive substrate analogs support
the idea of a common hydrophobic pocket within Pgp and show that residues from
multiple TM segments contribute to the binding region. 131-134 Cys cross-linking ex-
periments showed that the packing of the TM segments of Pgp is altered when drugs
bind: in a different way for each substrate. 135 This “induced-fit” type of mechanism
can explain how the binding pocket accommodates such a broad range of structurally
diverse compounds.
Like the transcriptional regulator proteins, the drug-binding pocket of Pgp appears
to be able to accommodate more than one compound simultaneously. Based on their
cross-linking data, Loo et al. proposed that a thiol-reactive substrate and a second drug
molecule could occupy different regions of the binding pocket simultaneously. 136
More recently, fluorescence approaches showed that LDS-751 and R123 could both
bind to the R-site of Pgp at the same time, interacting in a noncompetitive fashion. 137
The dimensions of the drug-binding pocket, determined using a thiol-reactive cross-
linking substrate, also suggest that it is large enough to accommodate two substrates
at the same time. 138
Several approaches have been used to locate and characterize the regions of Pgp
that form the drug-binding pocket. Labeling of the protein with various photoactive
drug analogs, followed by chemical or proteolytic cleavage and identification of the
labeled peptides showed that several TM segments in both halves of Pgp were involved
in substrate-binding. 139-142 Different regions of the protein were labeled by different
drug analogs, suggesting that they did not all bind at exactly the same location.