Linliang Yu

AcrB Trimer Stability and Efflux Activity, Insight from Mutagenesis Studies

The multidrug transporter AcrB in Escherichia coli exists and functions as a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains poorly understood. In a previous study, we have shown that individual AcrB subunit is capable of folding independently, suggesting that trimerization of AcrB follows a three-stage pathway in which monomers first fold, and then assemble. Here we destabilized the AcrB trimer through mutating a single Pro (P223) in the protruding loop of AcrB, which drastically reduced the protein activity. We replaced P223 separately with five residues, including Ala, Val, Tyr, Asn, and Gly, and found that AcrBP223G was the least active. Detailed characterization of AcrBP223G revealed that the protein existed as a well-folded monomer after purification, but formed a trimer in vivo. The function of the mutant could be partly restored through strengthening the stability of the trimer using an inter-subunit disulfide bond. Our results also suggested that the protruding loop is well structured during AcrB assembly with P223 served as a “wedge” close to the tip to stabilize the AcrB trimer structure. When this wedge is disrupted, the stability of the trimer is reduced, accompanied by a decrease of drug efflux activity.

Using U-Shaped Localized Surface Plasmon Resonance Sensors to Compensate for Nonspecific Interactions

We report a simple label-free localized surface plasmon resonance sensor that uses the multiple resonances of a U-shaped gold nanostructure to differentiate the target interaction of interest from background refractive index interference and nonspecific binding of interfering molecules. U-shaped nanostructures were fabricated using electron-beam lithography and their sensing capabilities were tested by introducing different solutions to simulate various specific and nonspecific effects. The three resonances of the nanostructure yield distinct bulk as well as specific and nonspecific surface sensitivities that allow for the differentiation of the three effects.

Characterization of the ligand and DNA binding properties of a putative archaeal regulator ST1710.

While a rich collection of bacterium-like regulating proteins has been identified in the archaeal genome, few of them have been studied at the molecular level. In this study, we characterized the ligand and DNA binding properties of a putative regulator ST1710 from the archaeon Sulfolobus tokodaii. ST1710 is homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators. The protein consists of a ligand binding site, partially overlapping with a winged helix-turn-helix DNA binding site. We characterized the interactions between ST1710 and three ligands, salicylate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and ethidium, which bind to bacterial MarRs. The binding affinities of the ligands for ST1710 were comparable to their affinities for the bacterial MarRs. The ligand binding was temperature sensitive and caused conformational changes in ST1710. To investigate the effect of ligand binding on the interaction between ST1710 and DNA, we fluorescently labeled a 47mer dsDNA (ST1) containing a putative ST1710 recognition site and determined the dissociation constant between ST1 and ST1710 using the fluorescence polarization method. The binding affinity almost doubled from 10 degrees C (Kd = 618 +/- 34 nM) to 30 degreesC (Kd = 334 +/- 15 nM), and again from 30 to 50 degrees C (Kd = 189 +/- 9 nM). This result suggests that under the natural living condition (80 degrees C) of S. tokodaii, the binding affinity might increase even further. The presence of CCCP and salicylate suppressed ST1710-ST1 interaction, indicating that ST1710 functioned as a repressor.

Differentiating surface and bulk interactions using localized surface plasmon resonances of gold nanorods

We demonstrate a novel localized surface-plasmon resonance sensor that can distinguish surface binding interactions from interfering bulk effects. This is accomplished by utilizing the longitudinal and transverse plasmon modes of gold nanorods. We have investigated, both numerically and experimentally, the effect of change in background refractive index and surface binding on the two resonances of a gold nanorod on an indium tin oxide coated glass substrate.

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.

Dissecting the function of a protruding loop in AcrB trimerization

The resistance-nodulation-cell division family multidrug transporter Acriflavine resistance protein B (AcrB) from Eschericha coli is an obligate homotrimer. Approximately, 45% of the inter-subunit interface is contributed by a protruding loop (also referred to as the thumb) and its corresponding binding tunnel in the neighboring subunit. In an earlier study, we have demonstrated that a single Pro to Gly mutation in the loop drastically destabilized AcrB trimer and reduced its substrate efflux activity. To further dissect the role of the loop during AcrB trimerization, we performed Ala scanning of the loop and examined the effect of each mutation on protein activity. We found that not all conserved residues are important for AcrB function and likewise not all critical residues are conserved. In addition, we replaced the loop of AcrB with the loop of MexB, which is a highly conserved homolog of AcrB. The resultant chimeric protein remained partly active. Structural characterization of the chimeric protein indicated that it was well folded and existed as a mixture of monomer and trimer. Our results indicate that the loop to tunnel interaction, while critical to trimerization and efflux function, is in general rather flexible and tolerant to mutations. In addition, all mutations in the loop that resulted in reduced function clustered closely, suggesting that this may be the site of inter-subunit recognition during trimerization and/or a locking zone to stabilize the inter-subunit interaction during trimerization.

Multi-mode localized surface plasmon resonance sensors for compensation of interfering effects

We demonstrate a novel localized surface plasmon resonance based sensor that can differentiate a target interaction from interfering interactions. This is accomplished by the simultaneous interrogation of the sensing medium with the multiple surface plasmon modes of the U-shaped gold nanostructure.