Acriflavine
resistance proteins B (PBD ID: 4ZIT) from Escherichia coli
Created by:
Negin Ghafourian
Bacteria can be exposed to foreign substances and
cytotoxic compounds, including clinical drugs. Pathogens overcome unfavorable
toxic environments for their survival by preventing drug access to the target
molecule. In gram negative bacteria, such as Escherichia coli, the
overproduction of the multidrug efflux pumps of the resistance nodulation
division (RND) protein superfamily is responsible for transferring these
noxious substrates out of the cell (11). Acriflavine resistance
proteins A/B-Tolerance to colicins (AcrAB-TolC) is one type of the RND with the
highest affinity for substrates. AcrAB-TolC consists of three
different protein components: the outer membrane efflux duct TolC, the inner
membrane-anchored adaptor protein AcrA and the inner membrane transporter
protein AcrB (PBD ID: 4ZIT) (1). Although all
three proteins are critical for drug efflux, AcrB utilizes the energy of a
proton gradient over the inner membrane and drives cyclic conformational
changes required to transport substrate from the periplasmic space out of
the cell (1).
AcrB has a total molecular weight of 681351.80 Da
and an isoelectric point of 5.39 (4). AcrB is homotrimeric, consisting of three
identical asymmetric chains with the same primary structure
(13). AcrB is comprised of three domains, each parallel to the inner membrane
and with different functions and structures: the transmembrane domain, the
porter domain and the docking domain. The extensive periplasmic portion of the
AcrB consists of porter and docking domains (8).
Proton conduction takes place in 12-helix transmembrane domain (2). The funnel-like
docking domain is responsible for binding the AcrB to the TolC part of the
efflux and transporting the substrate out of the cell. The porter
domain, responsible for substrate recruitment and transport, is composed of the
central helix bundle which consists of alpha helices, one from each promoter
(8). The porter domain is divided into four different subdomains: PN1,
PN2, PC1, and PC2, each having the beta-alpha-beta sandwich structure (8,2).
PN1 is located close to the core, while the other three subdomains are located
around the core (1). PN1 and PN2 are located at the N
terminus halfway between two transmembrane helices (TMH1 and TMH2),
and PC1 and PC2 are located halfway between TMH7 and TMH8. These four
subdomains pack together and form “proximal/access” and “distal/deep” substrate
binding pockets (1, 2).
According to crystallographic studies of AcrB,
large antibiotics, such as erythromycin, rifampicin or doxorubicin dimers,
mostly interact with the proximal pocket, while smaller substrates, such as
doxorubicin and minocycline, bind the distal pocket (1). Antibiotics such as
erythromycin undergo a sequential export process. First, the antibiotics
bind to the proximal pocket. Then they pass through the gate loop to the distal
pocket and subsequently extrude out of the cell via TolC (1). AcrB
loses its molecular symmetry when the drug binds to its promoters, adopting
distinct conformational states known as loose/access, tight/binding and
open/extrusion (7). Entrances and exits of the portal domain (PDe and
PDx respectively) are trapped in monomer-specific states of substrate
accessibility. In AcrB, monomers A and B, proposing loose/access and tight/
binding, exhibit open PDe/proximal binding pocket and close PDx. In monomer C,
the open/extrusion reaction cycle intermediate, PDe is closed, while PDx is
open. A small channel in the gap between PC1 and PC2 in the access conformation
state allows entrance of the substrate from the outer membrane to the vestibule
(3, 7). In the binding conformation, the cleft shrinks and the binding
pocket expands, thereby allowing the substrate to pass from the vestibule to
the expanded binding pocket. At the same time, the PN2 changes, causing a hydrophobic
pocket at the interface of the PN1 and PC1 (3, 7). The central
helix promoter blocks the exit, preventing the drug from exiting in this state
(7). However, during the extrusion state, the shrinking block pushes the drug
out (12).
The key feature of the AcrB is the short stretch
of residues known as the gate/switch loop, separating the proximal and distal
pockets. The gate loop consists of residue 615-620:FGFAGR (1). According
to multiple studies, mutation or deletion of any of these 5 residues can
negatively affect the transport process. Crystallographic data suggests that
the gate loop is involved in the substrate binding process since the central
phenylalanine, Phe-615, in gate loop interacts with erythromycin, which was
previously bound to the proximal binding pocket (1). This
contact corresponds to 10 percent of the antibiotic surface area. Removal or
substitution of the phenylalanine side chain significantly affects efflux
activity (1). There are two AcrB gate loop variants. In the first one, the AcrB
lacks the bulky side of the gate loop. In the second one, the protein does not
have a gate loop. According to experiments on these two variants, the lack of
phenylalanine does not lead to lower occupancy of erythromycin relative to the
wild type AcrB. The two variants’ ability to confer erythromycin tolerance is
significantly impaired. Although lacking a gate loop affects the
erythromycin-export mechanism, the binding of erythromycin to the proximal
binding pocket does not depend on the presence of a gate loop. The gate
loop substitution does not affect the overall folding pattern of the AcrB since
the two variants fold similarly compared to the wild type. Furthermore, their
trimeric state, or functional asymmetry, are the same (1).
In addition to gate loop significant residues, the residues Asp-407, Asp-408, Lys-940 and Thr-978 in the porter domain are important in the proton pathway. The Thr-978 hydrogen bonds with Asp-407, Asp-408 and Lys-940, creating a salt bridge. Altering any of these four amino acids leads to domain conformation changes and affects the transportation of the substrate (12).
The result of site-directed mutagenesis
determines another important residue, Arg-780, at
the trimer interface, which plays important roles in AcrB function. All Arg-780 mutants
are non-functional except the Arg-780-Lys Arg-780-Lys, which is partially
functional. Random mutagenesis identifies the repressor mutation, Met-774-Lys, restoring
the activity of AcrBR780A to a
similar level as wild type AcrB (6). The Arg-780 and Met-774 are
in close proximity in the crystal structure, suggesting the importance of the
positive charge in arginine in this location. According to these results,
the Arg-780 is an important functional residue critical
for stability. For instance, replacing the arginine with alanine does not
affect the overall structure of the protein, and the protein trimerizes
normally in the cell membrane. However, there are local structure
rearrangements that lead the protein to lose its substrate efflux activity (6).
AcrB consists of two ligands: Dodecyl-Beta-D-Maltoside (LMT),
nickel (II) ion (NI) (1). The protein sequence from Escherichia coli reveals
that the residues that are involved in ligand bindings are approximately 6 Å
or less from their ligands, which leads to the formation of hydrophobic, Van
der Waals, or electrostatic interactions. LMT involves in crystallization
of AcrB (1). At least one crystal contact appears to result from the presence
of an electron-dense metal ion, most likely ascribable to a
nickel ion carried over during affinity purification (1).
CusB (PBD
ID:3NE5) is part of the CusBA-heavy metal efflux complex in Escherichia
coli, and has a similar sequence to AcrB with an E value of 3e-50 and a Z
score of 42.9 (5). The Z score compares the tertiary structure of the two comparison protein; however, the E value
compares and describes the chance of not getting the similar sequence by random
chance (3, 10). CusBA-heavy metal efflux transfers ions such as Copper, and
Silver out of the cell; while the AcrAB efflux pump expels toxic compounds such
as antibiotics out of the cell. CusB contains 379 amino acids, while the AcrB
contains 1044 amino acids’ residues. CusB comprises three beta domains which
are mostly beta strands, and one domain which is alpha helices, folded into
alpha-helix bundle structure. Similar to trimeric structure of AcrB, hexametric
arrangement of CusB allows it to form funnel-like structure in the membrane. At
N terminus of the CusB, alpha helices, and random coil form. Three methionine
residues at the N terminus comprises three methionine binding site, helping to
transfer metal into periplasmic portion of CusA.
Despite their similar sequences, CusB and AcrB have 6 and 3
subunits, respectively. On the other hand, CusA, which is a part of the CusBA,
has similar functions to AcrB even though its sequence is not similar to each
other (5, 9, 15).
Escherichia coli protect themselves
from toxins and metal by forming and activating different triple efflux
complexes. When bacteria are exposed to erythromycin, AcrAB complex expels it
from the cell membrane and protects the bacterial cell. AcrB is the protein in
AcrAB complex responsible for separating proximal and distal binding pockets.
AcrB changes its conformation, which leads to three different states: loose/access,
tight/binding and open/extrusion. When the bacterial cell is exposed to ions
such as silver ions, the similar efflux pump, CusBA, transfers the metal out of
the cell membrane. CusB have similar tertiary and secondary structure to AcrB,
while they have different primary structures.