compared with that of the drug-free con-
formation, CH1–CH2 distance is symmet-
rically increased in the cryo-EM structures
(fig. S11G). Because the closed asymmetric
conformation in crystal structures is not ob-
served in any round of 3D classification (figs.
S9E and S10A) orcryoSPARC(Structura Bio-
technology, Canada) variability analysis (fig.
S12A) ( 35 ) of cryo-EM images, we posit that
the open C2-symmetric state is the predom-
inant conformation of G247-bound MsbA in
solution.
Comparing the cryo-EM structures of G247-
bound and drug-free MsbA ( 6 ) reveals a mech-
anism of G compound inhibition that is
different from previously anticipated: Instead
of pushing one NBD toward the dimerization
interface by 10 to 15 Å ( 25 ), G247 binding
displaces the NBDs symmetrically and away
from each other, which increases the inter-
NBD distance by ~13 Å (Fig. 4C). This model
is consistent with 3D variability analysis (fig.
S12A), in which G247-bound MsbA NBDs move
away from the dimerization axis relative to the
NBDs of drug-free MsbA. Whereas drug-free
MsbA has NBDs aligned in a head-to-tail man-
ner and thus primed for ATP hydrolysis, the
increased inter-NBD distance in G247-bound
MsbA is expected to reduce NBD dimerization
efficiency and ATPase activity. To formally test
this hypothesis, we exposed MsbA to the non-
hydrolyzable nucleotide AMP-PNP in the pres-
ence or absence of G247 (fig. S12B). When
subjected to 1 mM AMP-PNP, nearly all MsbA
particles showed a closed conformation with
dimerized NBDs. By contrast, 5mMofG247
was sufficient to shift >60% of particles to
an open conformation with separate NBDs
(fig. S12B).
Discussion
The effect on MsbA conformation and the
inhibition mechanism of TBT1 (fig. S13) are
different from those of small-molecule modu-
lators for other ABC transporters, which typ-
ically induce more modest conformational
changes (for example, ABCB1 and ABCG2 in
fig. S6D). The narrowed inter-NBD spacing of
TBT1-bound MsbA is more akin to the NBD
tightening that is observed when the human
multidrug transporter ABCC1 is exposed to its
endogenous leukotriene substrate (fig. S6D)
( 1 ), which further suggests that TBT1 trig-
gers substrate recognition–like behavior in
MsbA by mimicking the much larger LPS
substrate. Yet, unlike the authentic ligand,
TBT1 breaks the symmetry of homodimeric
MsbA by destabilizing one domain-swapping
helix bundle. Uncoupling of ATPase func-
tion and substrate transport by TBT1 occurs
through the domain-swapped CH, which is a
landmark feature of the type IV exporter fold
( 33 ). Thus, small-molecule decouplers acting
like TBT1 may be identified for many other
type IV exporters such as ABCB1 and ABCC1.
We have shown that G247, in contrast to
TBT1, suppresses ATPase activity by increas-
ing inter-NBD distance (fig. S13). This inhibi-
tion mechanism is similar to that of previously
described ABC transporter inhibitors, includ-
ing zosuquidar, which reduces the activity of
the homologous ABCB1 transporter by pushing
its NBDs away from each other ( 17 ). Because
most ABC transporter inhibitors character-
ized so far are ATPase suppressors, it is likely
that more compounds act similarly to G247
than to TBT1.
TBT1 and G247 currently face severe draw-
backs that prevent their application in clin-
ical settings, including low binding affinity
to MsbA ( 28 ) and interaction with animal
serum ( 29 ). Yet compound-bound ABC trans-
porter structures provide essential templates
for structure-based drug development be-
cause molecules such as TBT1 and G247 re-
veal unexpected binding pockets in previously
unidentified MsbA conformations. Our iden-
tification of W13 as an ATPase stimulator
would have been unlikely if the structure of
TBT1-bound MsbA were not available. One
promising direction for compound opti-
mization may consist of tethering together
two TBT1-like molecules to take advantage
of cooperative binding. Although TBT1 only
works onA. baumanniiMsbA, future efforts
may yield molecules that are able to extend
this inhibition mechanism to MsbA in other
Gram-negative bacteria, including additional
antibiotic-resistant pathogens.REFERENCES AND NOTES- Z. L. Johnson, J. Chen,Cell 168 , 1075–1085.e9 (2017).
- N. M. I. Tayloret al.,Nature 546 , 504–509 (2017).
- K. Nosolet al.,Proc. Natl. Acad. Sci. U.S.A. 117 , 26245– 26253
(2020). - Z. Zhang, J. Chen,Cell 167 , 1586–1597.e9 (2016).
- N. Liet al.,Cell 168 , 101–110.e10 (2017).
- W. Miet al.,Nature 549 , 233–237 (2017).
- H. Qianet al.,Cell 169 , 1228–1239.e10 (2017).
- J. A. Olsen, A. Alam, J. Kowal, B. Stieger, K. P. Locher,Nat.
Struct. Mol. Biol. 27 , 62–70 (2020). - D. C. Rees, E. Johnson, O. Lewinson,Nat. Rev. Mol. Cell Biol. 10 ,
218 – 227 (2009). - K. P. Locher,Nat. Struct. Mol. Biol. 23 , 487–493 (2016).
- C. Thomas, R. Tampé,Curr. Opin. Struct. Biol. 51 , 116– 128
(2018). - S. Srikant, R. Gaudet,Nat. Struct. Mol. Biol. 26 , 792– 801
(2019). - D. Szöllősi, D. Rose-Sperling, U. A. Hellmich, T. Stockner,
Biochim. Biophys. Acta Biomembr. 1860 , 818– 832
(2018). - S. Shukla, S. Ohnuma, S. V. Ambudkar,Curr. Drug Targets 12 ,
621 – 630 (2011). - A. M. Plummer, A. T. Culbertson, M. Liao,Annu. Rev. Physiol.
83 , 153–181 (2021). - V. G. Lewis, M. P. Ween, C. A. McDevitt,Protoplasma 249 ,
919 – 942 (2012). - A. Alam, J. Kowal, E. Broude, I. Roninson, K. P. Locher,Science
363 , 753–756 (2019). - A. Alamet al.,Proc. Natl. Acad. Sci. U.S.A. 115 , E1973–E1982
(2018). - S. M. Jacksonet al.,Nat. Struct. Mol. Biol. 25 , 333– 340
(2018). - B. J. Orlando, M. Liao,Nat. Commun. 11 , 2264 (2020).
21. C.F.Higgins,K.J.Linton,Science 293 , 1782– 1784
(2001).
22. A. Ward, C. L. Reyes, J. Yu, C. B. Roth, G. Chang,Proc. Natl.
Acad. Sci. U.S.A. 104 , 19005–19010 (2007).
23. W. T. Doerrler, M. C. Reedy, C. R. Raetz,J. Biol. Chem. 276 ,
11461 – 11464 (2001).
24. P. S. Padayattiet al.,Structure 27 , 1114–1123.e3 (2019).
25. H. Hoet al.,Nature 557 , 196–201 (2018).
26. G. Angiulliet al.,eLife 9 , e53530 (2020).
27. F. Thélot, B. J. Orlando, Y. Li, M. Liao,Curr. Opin. Struct. Biol.
63 , 26–33 (2020).
28. G. Zhanget al.,Proc. Natl. Acad. Sci. U.S.A. 115 , 6834– 6839
(2018).
29. M. K. Alexanderet al.,Antimicrob. Agents Chemother. 62 ,
e01142-18 (2018).
30. H. W. Boucheret al.,Clin. Infect. Dis. 48 ,1–12 (2009).
31. I.G.Denisov,S.G.Sligar,Chem. Rev. 117 , 4669– 4713
(2017).
32. M. A. McLean, M. C. Gregory, S. G. Sligar,Annu. Rev. Biophys.
47 , 107–124 (2018).
33. C. Thomaset al.,FEBS Lett. 594 , 3767–3775 (2020).
34. J. Wright, S. P. Muench, A. Goldman, A. Baker,Biochem. Soc. Trans.
46 , 1475–1484 (2018).
35. A. Punjani, D. J. Fleet,J. Struct. Biol. 213 , 107702
(2021).
ACKNOWLEDGMENTS
We are grateful to D. Kahne and D. Dates for providing the initial
TBT1 sample, as well as to Genentech for their donation of
G247. We thank J. Zhang for helping with ATPase screening
experiments. We thank Z. Li, S. Sterling, R. Walsh, and S. Rawson
from the Harvard Cryo-EM Center for Structural Biology for their
training and expert advice. TheA. baumanniiMsbA data and one
dataset ofE. coliMsbA-nanodiscs with G247 were collected at
the cryo-EM core facility at University of Massachusetts Medical
School, and the data of MsbA-DDM with G247 were acquired at
the Harvard Cryo-EM Center for Structural Biology at Harvard
Medical School. We are grateful to all Liao laboratory members for
their helpful feedback throughout this project.Funding:M.L.
was supported by an NIH grant from the NIGMS (R01 GM122797),
and J.H. was funded by the National Natural Science Foundation
of China (grant nos. 21803057 and 32171247).Author
contributions:M.L. conceived and supervised the project.
F.A.T. performed molecular cloning, protein purification, nanodisc
reconstitution, ATPase assays, negative-stain EM, sample
vitrification, cryo-EM data collection and processing, and model
building. K.S. and C.X. helped with cryo-EM data acquisition. W.Z
and J.H. performed virtual screen and molecular dynamics
simulation. F.A.T. and M.L. drafted the manuscript. All authors
contributed to data analysis and manuscript preparation.
Competing interests:The authors declare no competing financial
interests.Data and materials availability:Atomic models are
available through the Protein Data Bank (PDB) under the IDs 7MET
(TBT1-boundA. baumanniiMsbA), 7RIT (drug-freeA. baumannii
MsbA), and 7MEW (G247-boundE. coliMsbA in nanodisc).
Cryo-EM maps are available through the Electron Microscopy
Data Bank under the accession codes EMD-23803 (TBT1-bound
A. baumanniiMsbA), EMD-23804 (drug-freeA. baumanniiMsbA),
EMD-23805 (G247-boundE. coliMsbA in nanodisc), EMD-24446
(G247-boundE. coliMsbA in DDM, C2), and EMD-24447
(G247-boundE. coliMsbA in DDM, C1). Unprocessed motion-
corrected micrographs for all datasets are accessible through
EMPIAR under the accession codes 10778 (TBT1-bound
A. baumanniiMsbA), 10801 (drug-freeA. baumanniiMsbA), 10799
(G247-boundE. coliMsbA in nanodisc), and 10800 (G247-bound
E. coliMsbA in DDM). All other data are present in the main text or
supplementary materials.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abi9009
Materials and Methods
Figs. S1 to S13
Tables S1 and S2
References ( 36 – 57 )
MDAR Reproducibility Checklist11 April 2021; accepted 14 September 2021
Published online 23 September 2021
10.1126/science.abi9009SCIENCEscience.org 29 OCTOBER 2021•VOL 374 ISSUE 6567 585
RESEARCH | RESEARCH ARTICLES