Meng Zhong

Folding of AcrB Subunit Precedes Trimerization

AcrB and its homologues are major players in the efflux of anti-microbials out of Gram-negative bacteria. The structural and functional unit of AcrB is a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains elusive. It is not clear if an individual subunit folds into a monomeric form first followed by association (three-stage pathway) or if association occurs simultaneously with subunit folding (two-stage pathway). To answer this question, we investigated the feasibility of creating a folded monomeric AcrB mutant. The existence of well-folded monomers in the cell membrane would be an evidence of a three-stage pathway. A monomeric AcrB mutant, AcrB(Δloop), was created through the truncation of a protruding loop that appeared to contribute to the stability of an AcrB trimer. AcrB(Δloop) expressed at a level similar to that of wild-type AcrB. The secondary structure content and tertiary conformation of AcrB(Δloop) were very similar to those of wild-type AcrB. However, when expressed in an acrB-deficient strain, AcrB(Δloop) failed to complement its defect in drug efflux. Results from blue native polyacrylamide gel electrophoresis and chemical cross-linking experiments suggested that AcrB(Δloop) existed as a monomer. The expression of this monomeric mutant in a wild-type Escherichia coli strain did not have a significant dominant-negative effect, suggesting that the mutant could not effectively co-assemble with genomic AcrB. AcrB(Δloop) is the first monomeric mutant reported for the intrinsically trimeric AcrB. The structural characterization results of this mutant suggest that the oligomerization of AcrB occurs through a three-stage pathway involving folded monomers.

A Reporter Platform for the Monitoring of In Vivo Conformational Changes in AcrB

AcrB is an inner membrane protein in Escherichia coli that is a component of a triplex AcrA-AcrB-TolC (AcrAB-TolC) multidrug efflux pump. The AcrAB-TolC complex and its homologues are highly conserved among Gram-negative bacteria and are major players in conferring multidrug resistance (MDR) in many pathogens. In this study we developed a disulfide trapping method that may reveal AcrB conformational changes under the native condition in the cell membrane. Specifically, we created seven disulfide bond pairs in the periplasmic domain of AcrB, which can be used as probes to determine local conformational changes. We have developed a rigorous protocol to measure the extent of disulfide bond formation in membrane proteins under the native condition. The rigorousness of the method was verified through examining the extent of disulfide bond formation in Cys pairs separated by different distances. The blocking-reducing-labeling scheme combined with fluorescence labeling made the current method convenient, reliable, and quantitative.

Site specific and reversible protein immobilization facilitated by a DNA binding fusion tag.

Due to the complexity and diversity of protein structures, site-specific protein immobilization has always been challenging. On the contrary, DNA immobilization is straightforward with well established chemical methods. Single-strand DNA binding protein (SSB) binds tightly to single-stranded DNA (ssDNA). Herein, we investigated the feasibility of using SSB as a fusion tag to facilitate site-specific and reversible immobilization of target proteins. As a model system, we constructed a fusion protein by joining a superfolder green fluorescent protein (sfGFP) with SSB. The fluorescence emission and ssDNA binding affinity of the fusion protein were compared separately with those of the individual modules. Both modules fully retained their properties in the fusion construct. Next, we covalently attached ssDNA (dT(37)) to a supporting matrix through either amine or thiol functionalization. The attached ssDNA mediated reversible sfGFP-SSB immobilization. The immobilized protein could be released through changes of conditions including pH, concentration of divalent cations, and the presence of the complementary dA(35) oligonucleotide.

Insights into the Function and Structural Flexibility of the Periplasmic Molecular Chaperone SurA

SurA is the primary periplasmic molecular chaperone that facilitates the folding and assembling of outer membrane proteins (OMPs) in Gram-negative bacteria. Deletion of the surA gene in Escherichia coli leads to a decrease in outer membrane density and an increase in bacterial drug susceptibility. Here, we conducted mutational studies on SurA to identify residues that are critical for function. One mutant, SurA(V37G), significantly reduced the activity of SurA. Further characterization indicated that SurA(V37G) was structurally similar to, but less stable than, the wild-type protein. The loss of activity in SurA(V37G) could be restored through the introduction of a pair of Cys residues and the subsequent formation of a disulfide bond. Inspired by this success, we created three additional SurA constructs, each containing a disulfide bond at different regions of the protein between two rigid secondary structural elements. The formation of disulfide bond in these mutants has no observable detrimental effect on protein activity, indicating that SurA does not undergo large-scale conformational change while performing its function.

Effect of crowding by Ficolls on OmpA and OmpT refolding and membrane insertion

Folding of outer membrane proteins (OMPs) has been studied extensively in vitro. However, most of these studies have been conducted in dilute buffer solution, which is different from the crowded environment in the cell periplasm, where the folding and membrane insertion of OMPs actually occur. Using OmpA and OmpT as model proteins and Ficoll 70 as the crowding agent, here we investigated the effect of the macromolecular crowding condition on OMP membrane insertion. We found that the presence of Ficoll 70 significantly slowed down the rate of membrane insertion of OmpA while had little effect on those of OmpT. To investigate if the soluble domain of OmpA slowed down membrane insertion in the presence of the crowding agent, we created a truncated OmpA construct that contains only the transmembrane domain (OmpA171). In the absence of crowding agent, OmpA171 refolded at a similar rate as OmpA, although with decreased efficiency. However, under the crowding condition, OmpA171 refolded significantly faster than OmpA. Our results suggest that the periplasmic domain slows down the rate, while improves the efficiency, of OmpA folding and membrane insertion under the crowding condition. Such an effect was not obvious when refolding was studied in buffer solution in the absence of crowding.

Functional Relevance of AcrB Trimerization in Pump Assembly and Substrate Binding

AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB.

Direct fluorescence polarization assay for the detection of glycopeptide antibiotics.

Glycopeptide antibiotics are widely used in the treatment of infections caused by Gram-positive bacteria. They inhibit the biosynthesis of the bacterial cell wall through binding to the D-alanyl-D-alanine (D-Ala-D-Ala) terminal peptide of the peptidoglycan precursor. Taking advantage of this highly specific interaction, we developed a direct fluorescence polarization based method for the detection of glycopeptide antibiotics. Briefly, we labeled the acetylated tripeptide Ac-L-Lys-D-Ala-D-Ala-OH with a fluorophore to create a peptide probe. Using three glycopeptide antibiotics, vancomycin, teicoplanin, and telavancin, as model compounds, we demonstrated that the fluorescence polarization of the peptide probe increased upon binding to antibiotics in a concentration dependent manner. The dissociation constants (K(d)) between the peptide probes and the antibiotics were consistent with those reported between free d-Ala-d-Ala and the antibiotics in the literature. The assay is highly reproducible and selective toward glycopeptide antibiotics. Its detection limit and work concentration range are 0.5 microM and 0.5-4 microM for vancomycin, 0.25 microM and 0.25-2 microM for teicoplanin, and 1 microM and 1-8 microM for telavancin. Furthermore, we compared our assay in parallel with a commercial fluorescence polarization immunoassay (FPIA) kit in detecting teicoplanin spiked in human blood samples. The accuracy and precision of the two methods are comparable. We expect our assay to be useful in both research and clinical laboratories.

Role of a Conserved Residue R780 in Escherichia coli Multidrug Transporter AcrB

Multidrug efflux pumps play important roles in bacteria drug resistance. A major multidrug efflux system in Gram-negative bacteria is composed of the inner membrane transporter AcrB, outer membrane protein channel TolC, and membrane fusion protein AcrA. These three proteins form a large complex that spans both layers of cell membranes and the periplasmic space. AcrB exists and functions as a homotrimer. To identify residues at the trimer interface that play important roles in AcrB function, we conducted site directed mutagenesis and discovered a key residue, R780. Although R780K was partially functional, all other R780 mutants tested were completely nonfunctional. Replacement of R780 by other residues disrupted trimer association. However, a decrease of trimer stability was not the lone cause for the observed loss of activity, because the activity loss could not be restored by strengthening trimer interaction. Using both heat and chemical denaturation methods, we found that the mutation decreased protein stability. Finally, we identified a repressor mutation, M774K, through random mutagenesis. It restored the activity of AcrBR780A to a level close to that of the wild-type protein. To examine the mechanism of activity restoration, we monitored denaturation of AcrBR780A/M774K and found that the repressor mutation improved protein stability. These results suggest that R780 is critical for AcrB stability. When R780 was replaced by Ala, the protein retained the overall structure, still trimerized in the cell membrane, and interacted with AcrA. However, local structural rearrangement might have occurred and lead to the decrease of protein stability and loss of substrate efflux activity.

Repressive mutations restore function-loss caused by the disruption of trimerization in Escherichia coli multidrug transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding.

Diverse sequences are functional at the C-terminus of the E. coli periplasmic chaperone SurA

SurA is a major periplasmic molecular chaperone in Escherichia coli and has been shown to assist the biogenesis of several outer membrane proteins. The C-terminal fragment of SurA folds into a short β-strand, which forms a small three-stranded anti-parallel β-sheet module with the N-terminal β-hairpin. We found that the length of the C-terminal fragment, rather than its exact amino acid composition, had a big impact on SurA function. To investigate the determinant factor of the C-terminal sequence, we created a library of SurA constructs randomized in the last 10 residues. We screened the library and randomly analyzed 19 constructs that displayed SurA activity. The C-termini of these constructs shared little sequence similarity, except that β-strand-forming residues were preferentially enriched. Three SurA constructs were expressed and purified for structural characterization. Circular dichroism and fluorescence spectroscopy analyses revealed that their structures were similar to the structure of the wild-type SurA. Our results suggest that for scaffolding purpose proteins may tolerate various sequences provided certain general requirements such as hydrophobicity and secondary structure propensity are satisfied. Furthermore, the sequence tolerance of SurA at the C-terminus indicates that this area is not likely to be involved in substrate binding.