stimulates MsbA ATPase activity despite abol-
ishing LPS transport ( 28 ); the other group, here
referred to as G compounds, blocks both ATP
hydrolysis and LPS transport ( 25 , 29 ). Although
these first-generation MsbA inhibitors repre-
sent an important breakthrough, their mech-
anisms of inhibition are not well understood.
Particularly, TBT1-induced decoupling of ATPase
activity from substrate transport is puzzling
because most known inhibitors suppress ATP
hydrolysis of ABC transporters in membrane
environment ( 12 ). We thus sought to under-
stand how TBT1 and G compounds exert op-
posite allosteric effects on ATP hydrolysis,
even though both block LPS transport.
TBT1-bound MsbA adopts an asymmetric,
collapsed inward-facing conformation
TBT1 was identified as an LPS transport in-
hibitor and MsbA ATPase stimulator in strains
from theAcinetobactergenus ( 28 ). We there-
fore sought to explore the mechanism of TBT1
inhibition inAcinetobacter baumannii, an
ESKAPE pathogen (forEnterococcus faecium,
Staphylococcus aureus,Klebsiella pneumoniae,
A. baumannii,Pseudomonas aeruginosa, and
Enterobacterspecies) responsible for antibiotic-
resistant infections in patients ( 30 ).A. baumannii
MsbA was expressed inEscherichia colicells,
purified in dodecyl maltoside (DDM), and recon-
stituted in palmitoyl-oleoyl-phosphatidylglycerol
nanodiscs (fig. S1, A to G) ( 31 , 32 ). The basal
ATPase activity ofA. baumanniiMsbA in
nanodiscs was ~1mmol ATP per minute per
milligram of MsbA (Fig. 1A), which is approx-
imately fourfold lower than that ofE. coli
MsbA ( 6 ). Exposure of MsbA to TBT1 resulted
in four- to sixfold stimulation in ATPase activ-
ity (figs. S1, D and G, and S7B), as well as a
dose-dependent increase in reaction rate inMichaelis-Menten kinetics (Fig. 1A). TBT1-
induced ATPase stimulation was much more
pronounced in nanodiscs than in DDM (fig.
S1G), which suggests that nanodiscs are a
more native-like system than detergent for
investigating TBT1 action on MsbA. We thus
used single-particle cryo-EM to determine the
structure of TBT1-bound MsbA in nanodiscs at
an overall resolution of 4.3 Å, with the TMDs
at 4.0-Å resolution showing sufficient side-
chain densities for model building (Fig. 1B
and figs. S2 and S3).
The conformation of TBT1-bound MsbA is
asymmetric and distinct from all previously
determined structures of type IV ABC transport-
ers, which are characterized by their domain-
swapped transmembrane helices (TMs) ( 33 ).
The domain-swapping TM4–TM5 bundle is
well resolved in chain A yet disordered in chain
B (Fig. 1B), which is also evident in unmaskedSCIENCEscience.org 29 OCTOBER 2021•VOL 374 ISSUE 6567 581
Fig. 2. Drug-free
A. baumanniiMsbA in
nanodiscs adopts a
wide inward-facing
conformation.(A) Cryo-EM
reconstruction (5.2-Å resolu-
tion) ofA. baumanniiMsbA
in the absence of ligand.
The unsharpened map
filtered at 8-Å resolution is
displayed as an outline
to show the nanodisc.
(B) Section through surface
representation and compar-
ison for three structures:
A. baumanniiMsbA, TBT1-
boundA. baumanniiMsbA,
andE. coliMsbA (PDB
ID: 5TV4) ( 6 ). (C) Top-
down view of the substrate
binding pockets. (D) Side
views of the NBDs. Ca
distances between ABC
signature motif serine (S)
and opposing Walker A
glycine (G) residues are
indicated as dashed lines,
with values rounded to the
nearest integer shown in
each panel. (E) Top view of
the NBDs, seen from the
TMDs. Cadistances
between NBDs are indi-
cated as in (D).RESEARCH | RESEARCH ARTICLES