Indian Journal of Biochemistry & Biophysics
Vol. 48, February 2011, pp. 7-13
Minireview
MsbA ATP-binding cassette (ABC) transporter of E. coli: Structure and possible
flippase mechanism
Gautam Kaul* and Gurulingappa Pattan
N. T. Lab-I, Department Biochemistry, National Dairy Research Institute, Karnal, India
Received 03 August 2010; revised 23 December 2010
ATP-binding cassette (ABC) transporters utilize the energy present in cellular ATP to drive the translocation of
structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria and eukaryotes. In bacteria,
these transporters are considered to be important virulence factors because they play role in nutrient uptake and in the
secretion of toxins. The advances in structural determination and functional analysis of bacterial transporters have greatly
increased our understanding of the mechanism of transport of these ABC transporters. Although progress in the field of
structural biology has been made with the prokaryotic family members, it is likely that eukaryotic transporters will utilize
the same mechanisms for translocation process. In this review, we summarize the function of the known MsbA ABC
transporters in E. coli and mechanistic insights from structural and possible flippase mechanism studies.
Keywords: Structure, MsbA, ATP-binding cassette transporter, ATP hydrolysis, Flip-flop, E. coli
Introduction
The envelope of E. coli and other Gram-negative
bacteria contains two distinct membranes - the inner
membrane (IM) and the outer membrane (OM)
separated by the periplasm, a hydrophilic
compartment that includes a layer of peptidoglycan1.
The OM is an asymmetric lipid bilayer with
phospholipids forming the inner leaflet and
lipopolysaccharides (LPS) the outer leaflet. It plays an
important role in nutrient uptake and provides the
organism with a remarkable permeability barrier
conferring resistance to a variety of detergents and
antibiotics2. The integral transmembrane proteins span
the IM (also known as cell membrane) with α-helical
transmembrane domains, whereas lipoproteins are
anchored to the membrane via an N-terminal lipid
modification and face towards the periplasm. The
peculiar permeability barrier properties that
distinguish the OM from the IM are mainly due to the
presence of LPS in its outer leaflet. LPS is a potent
stimulant of innate immune response. The lipid A
——————
*
Author for correspondence
Tel: 91-184-2259133, 2259115, 6534422
Fax: 91-184-2250042
E-mail:[email protected]
Abbrevations: ABC; ATP-binding cassette; IH; intracellular
helices; IM; inner membrane; LPS; lipopolysaccharide; MDR;
multi-drug resistance; MSD; membrane spanning domain; NBD;
nucleotide binding domain; OM; outer membrane; TMD;
transmembrane domain.
moiety of LPS (also known as endotoxin) is detected
by the TLR4/MD2 receptor of the mammalian innate
immune system3. To transport this lipid A molecule to
the OM, the E. coli MsbA a member of ATP-binding
cassette (ABC) superfamily is required4,5.
A model of protein motion coupling energy input
to work was inspired by crystallographic snapshots of
MsbA. Central to this model is a switch in the
accessibility of a transmembrane chamber, implicated
in substrate binding from an inward to an outwardfacing configuration6. Bacterial proteins are
synthesized in the cytoplasm and are submitted to
cellular sorting which brings them to different cellular
compartments. Some of these proteins remain in the
cytoplasm, whereas others are targeted out of
cytoplasm7. ABC transporters are integral membrane
proteins that utilize the energy of ATP hydrolysis to
translocate a wide variety of solutes across the
cellular membranes. These molecular pumps are
found in all phyla and form one of the largest of all
protein families8. Purified as well as reconstituted
LmrA, an ABC transporter from Lactococcus lactis
possesses phospholipid fippase activity in vitro that is
dependent on exogenous ATP, suggesting that ATP is
required by this ABC transporter9.
The completion of the E. coli genome sequence10
has permitted analysis of the complement of
genomically encoded ABC proteins. There are 48
ABC transporters in humans and a total of 80 ABC
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INDIAN J. BIOCHEM . BIOPHYS., VOL. 48, FEBRUARY 2011
proteins makes the largest paralogous family of
proteins in E. coli. These 80 proteins include 97 ABC
domains and are components of 69 independent
functional systems. The ABC domains are the energy
generating domains of multi-component membranebound transporters. Almost 5% of the genome is
occupied by the genes encoding these ABC
transporters11. The ABC family is sub-divided into 22
sub-families of prokaryotic importers, 24 sub-families
of prokaryotic exporters and 10 sub-families of
eukaryotic proteins. It is projected that the
information collected about E. coli MsbA-ABC
transporters would provide a good base for study and
comparison of structural and functional aspects in the
homologous proteins from other organisms.
In this review, the structure of nucleotide binding
domain (NBD), the intact structure of MsbA molecule
and the possible flipping mechanism of LPS flippase
are discussed.
Structure of ATP-binding cassette, NBD
ABC transporters are integral membrane proteins
that utilize the energy of ATP hydrolysis to
translocate a wide variety of solutes across the
cellular membranes. These transporters typically
contain two transmembrane domains (TMDs) and are
energized by a nucleotide-binding domain (NBD)
dimer that closes and opens during cycles of ATP
binding and hydrolysis12,13. Every ABC transporter
share these common architectural organizations: two
hydrophilic NBDs located at the cytoplasmic surface
of the membrane and two hydrophobic TMDs or
membrane spanning domains (MSDs) that form the
translocation pathway. In prokaryotes, these domains
are mostly expressed as separate protein subunits,
whereas in eukaryotes they are usually fused into a
single polypeptide8.
Nucleotide binding subunits (also known as ATPbinding cassettes) power the transporter by binding and
hydrolyzing ATP. It is presumed that ATP binding and
or hydrolysis in the NBDs are coupled to
conformational changes in MSDs that moderate
unidirectional pumping of substrates across the
membrane14. It has been observed that four short motifs
are invariably conserved when multiple alignment of
primary sequence of the 94 ABC domains of E. coli
was analyzed11. In addition, sequence similarity is
observed throughout the rest of the ~215 amino acids
that make up the ABC domain15,16.
Although there is a large diversity of the transport
substrates, sequences of ABC components are
remarkably conserved among all ABC transporters.
The various conserved motifs of the NBDs of ABC
transporters are: Walker A motif, Q loop or the lid,
LSGGQ motif or the signature motif, Walker B motif
and H motif or the switch region. While the Walker A
(GXXGXGKS/T with X representing any residue)
and Walker B (ΦΦΦΦD with Φ representing any
hydrophobic residue) motifs are found in nearly all
ATP-binding proteins17-19, the signature motif
LSGGQ as well as the histidine (H) and glutamine
(Q) loop are unique to ABC transporters20.
The structure of NBD monomer can be divided into
two sub-domains: a larger Rec A-like21 (Fig. 1), which
consists of two β sheets and six α-helices and a
smaller helical sub-domain that is specific to ABC
transporters and is not seen in other ATPases14. In the
Fig. 1, ATP molecule is shown bound to the larger
RecA-like sub-domain and the γ (P) is positioned
close to the edge of one of β sheets, where it interacts
with several residues directly or via H2O. Other two
conserved motifs Walker A and B motifs are directly
involved in the binding and hydrolysis of ATP16. The
Q loop or the lid or the γ-phosphate switch22, which
joins the two sub- domains i.e., RecA-like subdomain and helical sub-domains of NBD monomer
appears to be highly flexible. The H motif is
conserved histidine located 20 amino acids
downstream of aspartic acid of Walker B motif11 that
forms a hydrogen bond with γ-(P) of ATP. The
Fig. 1—A homodimer structure of NBD of E. coli MalK (an E. coli.
maltose transporter) [Different conserved motifs are shown here.
RecA-like sub-domain (green), the helical subdomain (cyan),
Walker A motif (red), Walker B motif (blue), the signature motif,
LSGGQ, (magenta) and the Q loop (yellow) respectively. ATP
molecule is represented by ball and stick model. Molecule B of
homodimer has same color scheme as that of A, but are rendered in
lighter hue. (Modified and produced from Davidson and Chen14]
KAUL & PATTAN: E. COLI ABC TRANSPORTER MsbA
signature motif, also known as LSGGQ motif or
linker peptide has been used as the “signature” to
identify ABC transporters. It is the only major
conserved motif that does not contact nucleotide in
monomer structure and it also helps in the binding and
hydrolysis of ATP.
There is a dimeric arrangement of two NBDs in the
ABC transporters. The prediction was that NBD
would dimerize with ATP bound along the dimer
interface, flanked by the Walker A motif of one
subunit and the LSGGQ motif of the other. The
dimeric arrangement of the NBDs explains why all
ABC transporters have two ABC components, the
reason being that residues from both sub-units are
required to form the ATPase active site which is
located right at the dimer interface. It has been
difficult to determine the ATP requirement during the
transport i.e whether one or both ATPs are hydrolyzed
per transport event. In vivo measurements of growth
yields in bacteria suggest that only one ATP is needed
for the transport one substrate into the cell23. Unlike
prokaryotic NBDs, the NBDs of cystic fibrosis
transmembrane conductance regulator (CFTR), a
eukaryotic transporter are structurally asymmetric24, i.e.,
they contain two different NBDs: NBD1 and NBD2.
Structure of MsbA
MsbA is a 582 residue integral protein with six
putative transmembrane (TM) helices and one ABCtype NBD at its C-terminal end. A new AMP-PNPbound structure of MsbA has been solved to 3.7 Å
and shows a series of interacting helices that span the
bilayer and extend from the membrane into the
cytosol, where they are coupled to two interdigitated
NBDs25. MsbA is an essential ABC half-transporter
i.e., containing one TMD fused to a NBD, which
dimerize to form the full transporter in the
cytoplasmic membrane of E.coli. MsbA is essential
for cell viability and acts probably by mediating the
transport of lipid A, the hydrophobic moiety of LPS
from the cytoplasmic membrane to the OM26, serving
as anchor of LPS. Without a functional MsbA,
bacterial cell tends to accumulate a toxic amount of
lipid A within their inner membrane27, leading to cell
membrane instability and cell death. Furthermore,
when msbA gene is deleted from a capsule-deficient
strain of Neisseria meningitides28, an organism
that does not require LPS for viability29, this
msbA deletion mutant is viable with only a
slight growth defect and produces only small amounts
of phospholipids.
9
Spin-labeling electron paramagnetic resonance
spectroscopy has been used to determine the
conserved motifs within the MsbA NBD and it has
been found that LSGGQ NBD consensus sequence is
consistent with an α-helical conformation and,
therefore, these residues maintain extensive tertiary
contacts throughout hydrolysis25. MsbA is a member
of MDR (multi-drug resistance)-ABC transporter
group by sequence homology and is more closely
related to mammalin P-glycoproteins than any other
bacterial ABC transporter. Recently, it is reported that
purified MsbA binds various amphipathic drugs at a
location distinct from the site, where lipid A binds30.
MDR-ABC transporters are thought to function as
“hydrophobic vaccum cleaners”, because of their
ability to remove lipids and drugs from inner
membrane leaflet31. Also, MsbA transports
phosphatidylethanolamine (PE), indicating that it may
be a non-selective lipid transporter32.
The use of multi-copy refinement procedures and
analyzing the diffraction data using a single-copy
refinement procedure has helped to construct the
MsbA model33. Figure 2 shows three unique
conformations of MsbA which have the same
topology as Sav1866. The different conformations
reveal motions within the MsbA dimer, suggesting a
plausible mechanism by which accessibility switches
from inward to outward-facing. The three different
conformations of MsbA which help to transport
substrate from inward-to-outward are as follows:
Extracellular-facing conformation of MsbA with bound
nucleotide
There are six TM helices per monomer in the
structure of MsbA-AMPPNP. These TM helices
extend into the cytoplasm and interact with the NBDs,
hence as they provides the link between the site of
ATP hydrolysis and substrate transport pathway.
There are two short intracellular helix (IH) (Fig. 2A) :
(i) IH1 (residues 112-120) present in between TM2
and TM3 that inserts down into a groove above the
P-loop (378–386) of cis-NBD and makes contact with
the A-loop (351–358) and the nucleotide and (ii) IH2
(residues 212–220) between TM4 and TM5 and is
situated in a groove between the α- and β-subdomains
of trans-NBD. The helical TM6 provides physical
linkage between the TMD and NBD. Short extracellular loops (EL1; residues 54–59, EL2; residues
162–166, and EL3; residues 276–282) provide
connections between TM1/ TM2, TM3/TM4 and
TM5/TM6, respectively33.
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INDIAN J. BIOCHEM . BIOPHYS., VOL. 48, FEBRUARY 2011
Fig. 2—Three conformations of MsbA stereoviews [(A): Nucleotide bound; (B): open apo; and (C): closed apo. N-terminus is blue,
C terminus in red and the other in white. TM helices (TM1–TM6), extra-cellular loops (EL1–EL3) and intracellular helices (IH1–IH2) are
labeled accordingly. In the nucleotide bound state, the AMPPNP molecules are displayed. TM4/TM5/IH2 (yellow and orange) associates
with the opposite monomer in a conserved manner in all structures. Modified and produced from Ward and coworkers33]
Open inward (cytoplasm)-facing conformation of MsbA
A MsbA-open-apo of E. coli model has been built
by using MsbA-AMPPNP to study open inward facing
conformation of MsbA. The ATP-binding half sites
face each other, even though the NBDs are 50 Å apart
(Fig. 2B). The TM4/TM5/IH2 crosses over and
associates with the opposing monomer (Fig. 2B) and
this cross-over motif is highlighted by the open-apo
structure and provides the only means of interaction
between the two monomers. The cross-over interaction
buries 2,300 Å2 surface area per monomer and likely
holds the dimer together during the open inverted V
(inward-facing) conformation33. This type of
interlocking mechanism, known as an intertwined
interface is thought to reinforce stability and symmetry,
as seen in cytokines and DNA-binding proteins34.
Closed inward (cytoplasmic)-facing conformation of MsbA
A model of MsbA-closed-apo has been built with
the help of MsbA-open-apo and MsbA-AMPPNP
model as a guide. The verification concerned with
topology of the TMD has been carried out by mercury
positions Cys-88 and -315, and the placement of the
NBD in the electron density was confirmed by
mercury bound to Cys-401. This structure (Fig. 2 C)
represents
another
possible
inward-facing
conformation like MsbA-open-apo, i.e., without
nucleotide33.
Possible flipping mechanism
The term “flippase” was conceived to imply to
lipid transporters that serve to equilibrate newly
synthesized lipid across the biogenic membranes,
such as endoplasmic reticulum35. Several proteins
have been identified in eukaryotes that catalyze this
trans-bilayer movement of diverse class of lipids36,37.
But, MsbA is the only protein so far identified with a
proven role in lipid transport in prokaryotes. The
structure of MsbA implies a general mechanism for
hydrophobic substrate translocation by members of
the MDR-ABC transporter group. The transport of
amphipathic lipids across a hydrophobic lipid bilayer
was predicted to be thermodynamically unfavorable
and, therefore, lipid transporters demands energy
input38. The energy supplied will be utilized through
the stimulatory effect of purified hexa-acetylated lipid
A moiety and LPS on the ATPase activity of purified
and reconstituted MsbA proteins39 that perhaps
indicate the energy driven transport of lipid molecules
by the proteins2. The purified MsbA from E. coli
displays high ATPase activity and binds to lipids and
lipid-like molecules, including lipid A with affinity in
the low micromolar range40. A direct measurement of
the lipid flippase activity of purified MsbA in a
reconstituted system has been reported and the protein
displays maximal lipid flippase activity of 7.7 nmol of
lipid translocated per mg of protein over a 20 min period
for an acyl chain-labelled phosphatidylethanolamine
derivative41.
MsbA functions to transport LPS, which may
require significant conformational changes to move
the large sugar head groups across the membrane.
LPS is first synthesized on the inner leaflet and then
transported laterally across the inner membrane.
NBSs of Sav1866 and MsbA provide a snapshot of
the outward-facing conformation of an ABC exporter.
A comparison of this outward-facing conformation
KAUL & PATTAN: E. COLI ABC TRANSPORTER MsbA
with the inward-facing conformations highlights the
flexibility as well as the large range of motions
possible for this class of proteins. When the structure
of MsbA is analyzed, the open- and closed-apo
structures are inward-facing and these conformations
can accommodate substrate from either the inner
leaflet or the cytoplasm. The conformational changes
between the different structures of MsbA are most
apparent, when monomers from each state are aligned
by using transmembrane (TM) helices 1, 2, 3, and 6.
The comparison of the apo structures has shown
that TM4/TM5/IH2 can pivot ≈30° about a hinge
formed by EL2/EL3 in a rigid-body motion with
nearly conserved NBD alignment. This hinge creates
an inward-facing opening between TM3/TM6 and
TM4/TM5 in both apo conformations of MsbA.
Substrate binding to the inward-facing conformation
may promote the closure of the transporter’s TMDs,
which would in turn reposition the NBDs, allowing
the formation of the ATP sandwich in the presence of
nucleotide. The dimer interface in the inward-facing
conformations of MsbA is mediated exclusively by
the intertwined interface (TM4/TM5/IH2) with the
opposing monomer. In the absence of nucleotide, the
large buried surface area contributed by this interface
stabilizes the dimer, allowing flexibility in the
transporter while maintaining the relative orientation
of the individual domains33. The relative positioning
of TM4/TM5/IH2 is structurally preserved to the
opposing monomer in each conformation of MsbA
(Fig. 1)14. In the apo state, the NBDs cannot form the
proper interface required to achieve full catalytic
function, but the transporter may sample different
conformations.
In the nucleotide-bound state, the NBDs come
together to form a canonical ATP dimer sandwich,
significantly increasing the molecular interface within
the protein. This dimerization of the NBDs is coupled
to a packing reorganization of the TM helices relative to
the nucleotide-free state. The resultant twisting motion
pulls TM3/TM6 away from TM1/TM2 and causes a
change from an inward to an outward-facing
conformation33. In this ‘‘alternating access’’42 model of
MsbA, the inward and outward openings are mediated
by two different sets of TM helices (TM3/TM6 and
TM4/TM5 vs. TM3/TM6 and TM1/TM2, respectively).
The different openings may facilitate the unidirectional
transport of substrate across the membrane, especially if
the outward-facing conformation has lower affinity for
substrate43-45. At the dimer interface, MgATP-binding
11
residues and a network of charged residues are shown to
form a sequence of putative molecular switches that
allow ATP hydrolysis only at one nucleotide binding
sites (NBS)46.
Helical crystallization of MsbA and calcium
ATPases have also been recently carried out47 and the
conformational cycle48 of ABC exporter has been
worked out. It has further been reported that deleting
the two C-terminal alpha-helices is found to be
effective to crystallize the bacterial ABC transporter
E. coli MsbA which is complexed with AMP-PNP49.
The transition from nucleotide-free MsbA to the
highest energy intermediate changes distance in
liposomes which fit a simple pattern, whereby
residues on the cytoplasmic side (inward) undergo
20–30 Å closing motion, while a 7 to 10 Å opening
motion is observed on the extracellular side. The
transmembrane helices undergo relative movement to
create the outward opening50. The transport
mechanism in sav1866 involves an inward-facing
conformation with substrate binding site and outwardfacing conformation with an extrusion pocket exposed
to the external medium. The tight interaction of the
NBDs in the ATP bound state is coupled to the
outward facing conformation of the TMDs and bound
substrate will transported to the outer leaflet or
surrounding aqueous medium. The transporter is
returned to the inward-facing conformation due to the
ATP hydrolysis51.
Conclusion
The ABC proteins form the largest paralogous
family of proteins in E. coli. Various advances have
been made from the past several years in
understanding the molecular mechanism of
translocation in bacterial ABC transporters. Further,
there remains a lot to be learned about the process of
lipid movement in Gram-negative bacteria and the
true role of MsbA is not known clearly. Also, the
energy requirements need to be understood during the
process of transport. The structures of multi-drug
resistance (MDR)-ABC transporters can also help
elucidate the mechanism underlying the MDR
phenotype which could have a profound impact on the
development of novel therapeutics used to treat
cancer, infections and diseases. The complex range of
motion of a dynamic molecule can be understood with
the help of fluorescence resonance energy transfer
(FRET) and electron magnetic resonance (EPR) along
with the structural studies. The structure of MsbA and
mechanism of LPS transport needs to be further
12
INDIAN J. BIOCHEM . BIOPHYS., VOL. 48, FEBRUARY 2011
investigated. Till now lipid flippase has not been
purified in sufficient quantities to allow biochemical
characterization, While much has been put together
regarding the structure and molecular mechanism of
these ABC transporters in bacteria, many questions
remain stubbornly unanswered like the discrete
conformational changes in the NBD dimer that are
associated with the various stages of ATP hydrolysis,
complexity of coupling ATP hydrolysis, transport,
and peripheral proteins which will be a subject of
future research.
Acknowledgments
We thank Dr. Towseef Amin Rafeeqi and
Dr. Kamlesh Pawar for careful reading and insightful
comments of our manuscript. We acknowledge the
help of Mr Shashi Bhusan and Ms Sunita Kumari for
their assistance in the timely preparation of the
manuscript.
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