centralaxis(Fig.3C,bottom).TMD2thencol-
lapses around TM6.A, with TM2.A moving
closer to TM6.B. The convergence between
TM2.A and TM6.B might then push TM4–
TM5.B laterally. The unsolvable contradiction
between NBD.A moving toward the central
axis and TM4–TM5.B being pinched out-
ward results in the dissociation of CH2.B
from NBD.A.
TBT1 offers an example of a small-molecule
inhibitor that targets the central substrate-
binding site of a nonmultidrug ABC trans-
porter. Because most ABC transporters have
relatively narrow substrate spectra, our find-
ings suggest that mimicking substrate binding
can be a generally applicable strategy for de-
veloping small-molecule modulators for all
ABC transporters. To test whether the central
pocket is useful for structure-based compound
discovery, we performed an in silico chemical
library screen against the TBT1 induced-fit
pocket. Virtual screening of ~800,000 com-
pounds, followed by ATPase assay of select
hits, resulted in the identification of W13, a
compound that stimulated ATPase activity
with a median effective concentration lowerthan that of TBT1 (~5.5 versus 13mM) (Fig.
3F and fig. S8B). The same MsbA mutants
that were insensitive to TBT1 stimulation of
ATPase activity also demonstrated lower W13-
induced stimulation (fig. S7, B and C). W13 is
structurally dissimilar to TBT1 and consid-
erably larger in size (499 versus 336 Da) (fig.
S8A), and a single W13 molecule is expected
to occupy the central MsbA pocket (fig. S8, C
and D). Molecular dynamics simulation of
W13-bound MsbA in a POPC (1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine) lipid
bilayer suggests a binding mode that is rem-
iniscent of TBT1, including hydrophobic
groups of W13 extending upward in the hy-
drophobic pocket and carbonyl groups of
W13 near TBT1-recognizing arginine and
lysine residues (fig. S8D).G247 symmetrically increases inter-NBD
spacing and prevents MsbA closure
As the only other class of known MsbA-
specific inhibitors, G compounds are mecha-
nistically distinct from TBT1 because they
bind the inward-facing MsbA and prevent
ATP hydrolysis ( 25 ). The crystal structures
of G compound–boundE. coliMsbA in facial
amphiphile–3 (FA-3) detergent present asym-
metrical NBDs positioning ( 25 ), although how
the asymmetric conformation translates to
impaired conformational cycling remains un-
clear. Furthermore, the extraordinary confor-
mational flexibility of ABC transporters can
result in different states being captured, de-
pending on whether the protein is studied
with crystallography ( 21 , 22 , 24 ) or cryo-EM
( 6 , 26 ). We thus sought to further investi-
gate the mechanism of G compounds and
acquired cryo-EM datasets of G247-bound
E. coliMsbA in nanodisc (Fig. 4A and fig. S9)
and DDM (fig. S10).
In previous crystal structures of G compound–
bound MsbA ( 25 ), the NBDs are brought in
close proximity and positioned asymmetrical-
ly, with one NBD raised relative to the other
(Fig. 4B, right). By contrast, our cryo-EM struc-
tures in nanodisc and detergent are essentially
identical; both exhibit clear C2 symmetry,
even when refined without symmetry con-
straints (Fig. 4B, left, and fig. S11, B and C).
Asymmetry in the crystal structures stems
from Arg^190 , which forms a salt bridge with
the acrylic acid tail of the G compounds only in
one subunit of MsbA ( 25 ). In cryo-EM struc-
tures, Arg^190 is best ordered in the higher-
resolution DDM structure and appears too
distantfromG247toformasaltbridge(fig.
S11E). Furthermore, the TM4, TM5, and TM6
bundle is positioned differently relative to
TM1, TM2, and TM3 in the crystal and cryo-
EM structures (fig. S11F), which ultimately
affects the spacing between CHs (CH1 and
CH2) and NBD positioning. Whereas CH1–
CH2 spacing is reduced in crystal structures584 29 OCTOBER 2021•VOL 374 ISSUE 6567 science.orgSCIENCE
Fig. 4. G247 symmetrically increases inter-NBD spacing.(A) Cryo-EM reconstruction (3.9-Å resolution)
ofE. coliMsbA in complex with G247. The unsharpened map filtered at 6-Å resolution is displayed as an
outline to show the nanodisc. (B) Comparison of MsbA structures determined by cryo-EM or x-ray
crystallography (PDB ID: 6BPL) ( 25 ). The cryo-EM structure adopts C2 symmetry, whereas NBDs are
asymmetrically positioned in the crystal structure. (C) Comparison of the cryo-EM structures of drug-free
(PDB ID: 5TV4, gray) ( 6 ) and G247-bound (colored) MsbA, both in nanodiscs. G247 pushes NBDs away from
each other, compared with the uninhibited state. The NBD shift is apparent when viewing MsbA from
(left) the side and from (right) the cytoplasmic space. The dashed box in the MsbA side view (left) represents
the cross section of the NBDs shown at right.
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