RESEARCH ARTICLES
◥LIPID TRANSPORT
Distinct allosteric mechanisms of first-generation
MsbA inhibitors
François A. Thélot1,2, Wenyi Zhang3,4, KangKang Song5,6, Chen Xu5,6, Jing Huang3,4, Maofu Liao^1 *
ATP-binding cassette (ABC) transporters couple adenosine 5′-triphosphate (ATP) hydrolysis to substrate
transport across biological membranes. Although many are promising drug targets, their mechanisms of
modulation by small-molecule inhibitors remain largely unknown. Two first-generation inhibitors of the MsbA
transporter, tetrahydrobenzothiophene 1 (TBT1) and G247, induce opposite effects on ATP hydrolysis. Using
single-particle cryo–electron microscopy and functional assays, we show that TBT1 and G247 bind adjacent
yet separate pockets in the MsbA transmembrane domains. Two TBT1 molecules asymmetrically occupy the
substrate-binding site, which leads to a collapsed inward-facing conformation with decreased distance
between the nucleotide-binding domains (NBDs). By contrast, two G247 molecules symmetrically increase
NBD distance in a wide inward-open state of MsbA. The divergent mechanisms of action of these MsbA
inhibitors provide important insights into ABC transporter pharmacology.
A
TP-binding cassette (ABC) transporters
perform diverse functions that include
importing and exporting small mole-
cules, mediating ion channel opening,
and translocating lipids across cell mem-
branes ( 1 – 8 ). ABC transporters have two trans-
membrane domains (TMDs), which interact with
and transport substrates, and two nucleotide-
binding domains (NBDs), which bind and
hydrolyze adenosine 5′-triphosphate (ATP).
Decades of investigation have led to various
models to describe ABC transporter mecha-
nisms. In the alternating access model ( 9 – 13 ),
the substrate is first recognized by the inward-
facing transporter, subsequent ATP bind-
ing in the NBDs promotes a transition to the
outward-facing state and substrate release,
and last, ATP hydrolysis resets the trans-
porter in the inward-facing state. Interrupt-
ing such conformational transition cycles has
direct applications in treating cancer, regulat-
ing cholesterol homeostasis, and developing
antibiotics ( 14 – 16 ).
Small-molecule inhibitors have been devel-
oped against human multidrug ABC transporters,
including the ABCB1 inhibitors zosuquidar,
tariquidar, and elacridar and the ABCG2 in-
hibitors MZ29 and MB136 ( 3 , 17 – 19 ). Structu-
ral studies have revealed how these compounds
bind to the TMDs, which interrupts confor-
mational transition and consequently decreases
adenosine triphosphatase (ATPase) activity
( 3 , 19 , 20 ). By contrast, there is little structural
insight into small-molecule modulation of most
other ABC transporters, which demonstratenarrower substrate specificity. We do not know
whether modes of small-molecule inhibition
are shared across the ABC superfamily or
whether there are general druggable con-
formations or pockets. These knowledge gaps
limit our ability to rationally design drugs that
target many ABC transporters.
MsbA is a model system to study ABC trans-
porter mechanism ( 21 , 22 ). Because of its
essential role in lipopolysaccharide (LPS)
biogenesis in Gram-negative bacteria, MsbA
is also an attractive target for developing
antibiotics ( 23 ). X-ray crystallography and
cryo–electron microscopy (cryo-EM) studies
have determined MsbA structures in seve-
ral conformations, including ligand free, LPS
bound, adenylyl-imidodiphosphate (AMP-PNP)
bound, and vanadate trapped ( 6 , 22 , 24 – 26 ).
The structural basis of specific binding and
transport of LPS by MsbA has been elucidated
( 6 , 25 , 27 ). Compared with the recent rapid
progress in studying MsbA function, the un-
derstanding of MsbA inhibition has lagged
behind ( 27 ).
Two types of specific MsbA inhibitors have
been reported: One has a tetrahydrobenzothio-
phene (TBT) scaffold, named here as TBT1, andRESEARCH
580 29 OCTOBER 2021•VOL 374 ISSUE 6567 science.orgSCIENCE
(^1) Department of Cell Biology, Blavatnik Institute, Harvard Medical
School, Boston, MA, USA.^2 Biological and Biomedical Sciences
Program, Harvard University, Cambridge, MA, USA.^3 Key
Laboratory of Structural Biology of Zhejiang Province, Westlake
University, Hangzhou, China.^4 Westlake AI Therapeutics Lab,
Westlake Laboratory of Life Sciences and Biomedicine,
Hangzhou, China.^5 Department of Biochemistry and Molecular
Pharmacology, University of Massachusetts Medical School,
Worcester, MA, USA.^6 Cryo-EM Core Facility, University of
Massachusetts Medical School, Worcester, MA, USA.
*Corresponding author. Email: [email protected]
Fig. 1. TBT1 binding induces a collapsed inward-facing conformation of MsbA.(A) ATPase activity
ofA. baumanniiMsbA in nanodiscs, measured at varying ATP and TBT1 concentrations. TBT1 increases the
rate of ATP hydrolysis in a dose-dependent manner. Each point represents mean ± SD (calculated from
three independent measurements). (B) (Left) Cryo-EM map (4.0-Å resolution) ofA. baumanniiMsbA in complex
with TBT1, with domain-swapping TM4–TM5 colored fuchsia. The unsharpened map filtered at 10-Å resolution
is displayed as an outline to show the nanodisc and tightened NBD positioning. (Right) Cartoon of TBT1-bound
MsbA with structural elements indicated that illustrates the structural asymmetry of the two MsbA chains.
EH, elbow helix. A flipped cartoon can be found in fig. S4A. (C) Cryo-EM reconstruction low-pass filtered
at 6-Å resolution. The dashed box points to the N-terminal end of TM4.B, where TM4–TM5.B becomes
disordered and absent from the reconstruction. (D) Representative 2D class averages of TBT1-bound MsbA,
showing constricted TMDs yet separate NBDs. The box size is 203 Å.