Acriflavine Resistance Channel Protein B
Created by Mary Gasser
Acriflavine resistance channel protein B, or
AcrB protein (PDB ID: 2DRD), is a multidrug efflux protein that spans the length of the inner membrane and extrudes into the periplasmic region of the Gram-negative bacteria, Escherichia coli (1, 2). The theoretical pI of this protein is 5.55, and the theoretical molecular weight is 342,330.60 Daltons; these values were determined by the ExPASy Proteomics server. AcrB channel protein is a part of a three protein pump that also consists of proteins AcrA and TolC; this pump is the primary path by which E. coli expels various antibiotics, dyes, bile salts and detergents (3). With AcrA and TolC, AcrB can remove an assortment of toxic materials that include anionic, cationic, zwitterionic and neutral compounds (1). AcrB functions as the transporter mechanism of the pump; it is important for recognition of substrates and energy transduction, and it acts as a proton-drug antiporter (3, 4).
AcrB is important to the survival of E. coli because it removes antibiotics and other harmful chemicals from the organism that would otherwise be toxic. The tripartite AcrA/AcrB/TolC system is recognized as one of the most important of the multi-component efflux systems because of its ability to remove toxic substrates at high levels of efficiency when compared with other efflux transporters (4). The over-expression of this efflux system in E. coli is a major cause of the multidrug resistance phenotype, because of its ability to remove different classes of antibiotics (4). As part of a single efflux system, AcrB is able to confer resistance to E. coli to a wide range of different compounds that could decrease the chance of survival of the bacteria (3). AcrB also has serious ramifications for human health because of the antibiotic resistance it confers to E. coli. The multidrug resistance conferred by AcrB channel protein and its affiliate proteins is a serious medical concern, both regarding the chemotherapy of cancer and the antibiotic treatment of different bacterial infections (1).
AcrB protein has
three layers, each parallel to the inner membrane: the transmembrane domain, the porter domain and the TolC docking domain (1). The transmembrane domain anchors the protein in the inner membrane and plays an important role in proton translocation; it is composed of 12 transmembrane alpha-helices (1). The porter domain is one of the most significant in terms of contribution to the function of the AcrB protein. The central helix bundle is located in this domain; the bundle is composed of three alpha-helices, one from each protomer (1). The porter domain for each of the three protomers is composed of
four subdomains: PN1, PN2, PC1, and PC2. Each of these subdomains is composed of two beta-alpha-beta sandwiches (1). The PN1 subdomains are located closest to the core, while the other three subdomains of each protomer encompass the core (3). It is theorized that the PN1 subdomains enforce the order of conformational change (5). The TolC docking domain is the region of AcrB protein which is believed to bind to another protein in the tripartite complex, TolC. The structure of this domain is comparable to a funnel through which AcrB pumps substrates into TolC and out of the cell (1, 3).
AcrB protein is a
trimer, and its three chains have identical primary structures. The structures of the three chains are asymmetric: each has a different conformation in the porter domain (5). The different conformations of the protomers correspond with the three stages of the efflux cycle by which drugs and other harmful substances are removed; the three conformations are referred to as
access, binding, and extrusion (6). At any given time, all three of these conformations are present, and each of the three protomers has a different conformation. The access conformation gives potential substrates an
entrance to the vestibule from the outer leaflet of the membrane via a small channel, which forms at the gap between subunits PC1 and PC2 (3, 6). In the binding phase, the
cleft shinks slightly and the binding pocket expands so that the substrate passes from the vestibule to the expanded pocket; the PN2 subdomain is altered, which creates a hydrophobic pocket at the interface of the PN1 and PC1 subdomains (3, 6). The drug is not released during this phase because the central helix of the extrusion protomer blocks the exit (6). During the extrusion state, the drug is pushed out by the shrinking pocket; the
entrance is closed and the exit is open (6). Once a protomer has undergone the three conformational changes, the cycle repeats itself (2).
There are three particular ligands with which AcrB protein has been crystallized:
minocycline,
doxorubicin, and 9-bromo-minocycline (1). They were observed in one of the protomers in the binding pocket, located between the beta-sheets of the PC1 and PN2 subdomains (1). When located within the binding pocket, the methyl group of the 7-dimethylamino group of
minocycline, an antibiotic, interacts with F178, the C-ring interacts with F-615, and the oxygen atoms of the 1-oxo and 2-amido groups interact with N-274; 9-bromo-minocycline interacts analogously (1, 7).
Doxorubicin, a chemotherapeutic agent, interacts with F-615 similar to minocycline, but instead of N-274 and F-615, it interacts with Q-176 and F-617, respectively (1, 7). The
binding pocket has many aromatic residues, such as F-136 and F-178 in the PN2 subdomain, and F-610, F-615, F-617 and F-628 in the PC1 subdomain (1). There are polar residues such as N-274 and Q-176 and hydrophobic residues, including V-139, V-612, I-277, I-626, and Y-327 in binding pocket (1, 7). The variety of residues present suggests that
different residues within the binding pocket can bind different substrates; this versatility of the pocket contributes to the multidrug efflux capability of AcrB protein (1). Despite the data on residue and protein structure and function, there is a basic lack of understanding of drug binding within the protein (2).
There are several sets of residues within AcrB protein that are functionally significant. One such set whose interactions are important for structure and function are Q-112 and Q-108 (1). Q-112 residues in the binding and access protomers form a hydrogen bond; this causes the
inclination of the central alpha-helices for these protomers (1). The Q-112 residue of the extrusion protomer forms a hydrogen bond with Q108 of the binding protomer; this alters the inclination of the alpha-helix of the extrusion protomer (1). The altered alpha-helix plays a central role in blocking the substrate from being released before the protomer changes conformation from the binding to the extrusion state (1).
Another important group of residues plays a vital role in the proton translocation pathway in the transmembrane region: D-407, D-408, K-940, and T978 (5, 7). K-940 forms salt bridges with D-407 and D-408 in the
binding and
access states; the side chain of T978 also interacts closely with the three other residues (7). This network is disrupted in the
extrusion protomer, likely as a result of the protonation of D-407 (7). It is hypothesized that the protonation or deprotonation of one of the three residues of the salt bridge affects the ability of substrates to be bound or released from AcrB protein due to the structural changes in the transmembrane region that affect the movement of subdomains in the porter region (1, 6).
The mutant protein acriflavine resistance protein B (PDB ID: 2HQF) was chosen for comparison with AcrB channel protein based on similarity in tertiary structure. The Z-score was 48.3 and RMSD was 1.8 (8). The Z-score indicates similarity in tertiary structure between two proteins; it significant because it is above a value of 2 (8). The RMSD indicates the difference in angstroms between the
superimposed tertiary structures of the wild-type and mutant proteins (8).
Mutant acriflavine resistance protein B has a single mutation in the amino acid sequence when compared with the wild-type AcrB channel protein in which K-940 is changed to A-940; this mutation causes significant changes in the conformation of AcrB (9). Considering the important role that K-940 plays in the
salt bridge-hydrogen bonding network, the switch from a charged to a non-polar residue significantly changes the structure and functional ability of the protein, particularly in the salt bridge-hydrogen bonding network when
compared with the wild-type AcrB. One structural change observed was the alteration of the binding pocket size; this change may prevent the binding and efflux of larger drugs by the mutant protein (9). The study of the mutant AcrB protein indicates that the salt bridge-hydrogen bonding network of the four residues has considerable structural and functional ramifications for the entire AcrB protein. Changes in the structure of AcrB protein due to the mutation cause the
multidrug binding site in the wild-type AcrB protein to change in shape; it is hypothesized that the mutant AcrB protein may be unable to bind larger drugs as a result of this change (9). If it is the case that the mutant is unable to bind larger drugs, it is possible that E. coli containing the mutant protein would be less effective at removing certain drugs or other harmful substances, thereby decreasing the likelihood of survival of the organism.