coli TolC Multidrug Efflux Pump
Katie Sears '10 and Kristina Buschur '11
Gram-negative bacteria employ efflux pumps to expel
unwanted materials from their cytoplasm. Cells rely on this process for
survival. The pump is transient and consists of three proteins when it
is complexed. Two inner-membrane proteins make up the translocase
complex, which recognizes specific substrates and provides energy for
the expulsion step. When the translocase complex is bound to its export
substrate, it recruits a third, outer membrane protein known as TolC.
The complex is capable of expelling intruders in a single
energy-dependent step across both the outer and inner membranes and
across the periplasmic space between them. This is possible because
TolC stretches between the two membranes, creating a direct path from
the cytoplasm to the extracellular space (Koronakis et al.,
TolC has the capacity to interact with various
translocase complexes with different export substrates. Possible
substrates (each with their own translocase complex) include large
proteins like enzymes and toxins, as well as small agents like
detergents, solvents, heavy metals, and antibiotics. Expulsion of these
materials is vital to the survival of the cell.
The export of antibiotics by efflux pump is
particularly significant because it has been shown to contribute to
antibiotic resistance. There are two types of antibiotic resistance:
drug-specific and multidrug. Drug-specific antibiotic resistance is the
type we typically hear about, sometimes caused by the use of
antibiotics which place selective pressure on bacteria. This can lead
to advantageous mutations and new strains of bacteria, which are
resistant to particular substances. In these kinds of cases, mutations
encoding efflux systems have been observed on mobile genetic elements
like transposons and plasmids. On the other hand, multidrug-resistant
bacteria have efflux systems that are encoded chromosomally and
expressed constitutively. The endogenous nature of the multidrug efflux
proteins indicates that they have not evolved specifically in response
to antibiotic pressures (though they might be upregulated in response
to these pressures). These complexes have been around for longer
periods of time and are thought to have been intended to perform some
sort of housekeeping function. Though they may not have evolved for the
purpose of antibiotic export, these complexes have proven advantageous
for this challenge to cell survival. (Poole, 2007).
The study of multidrug efflux pumps could lead
to new treatments for human bacterial infection. Disruption of efflux
machinery could be accomplished directly, through interference of gene
or protein expression, or by inhibition of pump assembly (Poole, 2007).
For example, it has already been shown that loss of the TolC component
can result in lower bacterial survival rates (de Cristobal et
al., 2006), making this a possible mechanism of antibacterial
II. Overall Structure
The TolC protein is a trimer of
are each comprised of 428 residues. The overall shape of the protein is
that of a long tube, making it well-suited for channeling antibiotics
and other harmful molecules out of the bacterial cell.
long axis is
almost 140 Å long, and the cylinder structure has an internal
diameter of 35 Å.
of the three individual protomers consists of a
peptide chain that weaves up and down four times, forming three
distinct domains along the long axis of the molecule: a
β-barrel domain, an α-helical domain, and a mixed
α/β equatorial domain.
consists of four
β-sheets which appear at the distal (upper) end of the
is made up of 6
α-helices, two of which extend to the proximal (lower) end of
is two small β-sheets
and three small α-helices that form a band around the middle
of the helical barrel. A
of the protomer shows the
secondary structure elements, their position relative to one another,
and their location in the overall structure.
the trimer structure, the antiparallel β-sheets form a
12-stranded, right twisted β-barrel. The barrel is
approximately 40 Å long. In the cell, the β-barrel
end of the molecule faces the outer membrane.
form a 12-stranded antiparallel, left-twisted barrel with an internal
diameter of 35 Å and length 100 Å. The internal
space has a volume of approximately 43,000 Å3;
one of the largest ever discovered in a protein. In the cell, the
α-helical barrel extends into the periplasm.
end the structure is open, but at the proximal end it tapers almost to
the β-barrel and the α-helical barrel
meet, abrupt turns in proline-containing
accommodate the transition from a left-twisted barrel to a
right-twisted barrel. (Koronakis et al.,
membrane factors (OMFs) that have been characterized, the TolC OMF
β-barrel is wide open and accessible to molecules such as
order to maintain this conformation, the
strands must be twisted and curved. This is accomplished by the
placement of small or unbranched residues—including serine and
aspartic acid, which can pack closely with other
molecules—toward the inside of the barrel.
addition to giving the barrel its shape, these
small side chains contribute to the open conformation of the barrel. In
contrast, larger, more bulky residues have side chains outside of the
particular, the aromatic residues phenylalanine
and tyrosine form
clusters on the
outside of the barrel that define where TolC OMF meets the inner
edge of the outer E. coli outer membrane.
is a clear indication that the TolC protein is
positioned similarly to other OMFs that have been previously
characterized (Koronakis et al., 2000).
α-helical barrel extends into the periplasm of the
bacterial cell. The helices curve and twist around each other and
are considered coiled coils. They have a left-handed superhelical twist
throughout the entire barrel, but untwist somewhat near the distal end
of the barrel.
two long helices from each protomer (H3
remain uniform from the distal end of the barrel to where the
equatorial domain curves around the barrel. From that point till they
reach the proximal end of the barrel they begin to gradually taper in.
H7 and H8
form a conventional coiled coil in which both helices
the other hand,
H3 remains straight
coils around it. (Koronakis et al., 2000)
The coiled coils also interact with each other.
on H4 hydrogen bonds
on H7 and Gln136
on H3 hydrogen bonds with Glu359
(Andersen et al., 2002).
Many residues, such as these, that participate in hydrogen bonds
between the coiled coils
are highly conserved in the TolC family.
interacts with several inner membrane translocase complexes to be able
to export a large variety of materials from the cytoplasm. To export
molecules as large as whole proteins, the tapered proximal end of TolC
must be able to open and the proteins to be exported must be at least
is possible that this opening happens when the
coiled coils rotate slightly around the outer H3/H4
coils. Koronakis et al. propose that this small
change in the structure
of TolC could open the tunnel by as much as 30 Å (2000). They
also suggest that this change is induced by the recruitment of TolC by
likely that the
recognizes the translocase complex interaction because its
β-sheets and α-helices are arranged close to the
set of coiled coils and any change in their relative positions
could induce uncoiling of the helices.
TolC is recruited by a translocase that has bound a particular
substrate that is to be exported. This provides specificity for what is
exported and allowed access to the TolC tunnel. When TolC is complexed
with the translocase, a transient connection directly from the
cytoplasm, across the inner and outer membranes and periplasm, and to
the outside of the cell is established. There is a high degree of
sequence similarity among members of the TolC family, suggesting that
this proposed mechanism for transport could be common to all bacterial
efflux pumps. (Koronakis et al.,
protein of many
multidrug efflux pumps. The major drug efflux pump of E. coli
is the AcrA-AcrB-TolC complex.This transport protein complex is part of
the resistance nodulation cell division (RND) family—one of
five major classes of bacterial efflux systems (Poole, 2007).
is a periplasmic protein in the efflux pump complex. Here, the stable
core of AcrA was crystallized with four molecules per asymmetric unit.
One AcrA protomer looks like this:
The AcrA molecule is thought to be trimeric (a
complex of three of these protomers) in vivo, but
this has not been proven. Each protomer of this protein has three
domains: a β-barrel,
a lipoyl domain,
and a coiled-coil
belongs to the membrane fusion protein (MFP)
family, which includes proteins that also play a role in two other
efflux pump families: ATP-binding cassette (ABC) and major facilitator
system (MFS). Though it is not known precisely how AcrA interacts with
TolC and the third protein of the efflux
pump—AcrB—Mikolosko et al.
postulate that, in addition to helping maintain the formation of the
efflux pump complex, conformational changes in AcrA might also play a
role in TolC channel opening (2006).
spans the inner membrane. It is a trimer
consisting of three molecules that create a small channel.
three molecules cycle through three different
conformations in order to propel substrate molecules from the cytoplasm
toward the TolC OMF, to be expelled from the cell. The
conformational changes that make this molecule work are fueled by
proton-motive force within
the transmembrane domain. One end of the AcrB
molecule, oriented toward the cytoplasm, serves as an occlusion site
where substrate molecules enter the pump. This domain is made up mostly
other end of the molecule, located in the
periplasm, acts as the
TolC docking domain and is a complex structure of α-helicies and β-sheets.
docking domain is where the α-helical channel of TolC fits
into the AcrB inner membrane protein. Little is known about the
specific interactions between the two proteins at this time (Seeger et
C, Koronakis, E, Bokma, E, Eswaran, J, Humphreys, D, et al.
to the open state of the TolC periplasmic tunnel entrance.
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