细菌AcrAB-TolC型外排泵中AcrA蛋白的进化分析

研究不同耐药细菌AcrAB-Tolc型外排泵中关键蛋白AcrA的序列,针对其保守及非保守氨基酸残基进行该类蛋白的进化分析,构建蛋白进化树。收集来源于不同细菌的已知序列的AcrA蛋白,去除冗余并进行序列比对之后,根据其序列比对结果的相似性、氨基酸残基的保守性研究其进化特征。结果表明,不同细菌的AcrA蛋白部分氨基酸残基具有高度的保守性,这与其实现生物学功能有关,非保守区域是主要的进化区域。可为不同菌株的进化提供参考,同时为以AcrAB-Tolc型外排泵为靶标的新药研究提供相关数据。     

Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and β-barrel domains of AcrA.     

The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC

Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.AcrA, the multidrug efflux protein from Escherichia coli, is the best-characterized member of the membrane fusion protein (MFP) family (24). Periplasmic AcrA associates with the inner-membrane transporter AcrB, belonging to the RND superfamily of proteins, and the outer-membrane factor TolC (22, 23). Together, the three components form a transenvelope multidrug efflux pump responsible for the high levels of intrinsic as well as acquired antibiotic resistance of E. coli.AcrA is anchored into the inner membrane by N-terminal lipid modification. However, genetic complementation studies showed that the presence of the lipid moiety is not required for AcrA function (14, 24). Structural studies of the proteolytically stable core of AcrA (amino acid [aa] residues 46 to 312) and of whole-length MexA, a homologous protein from Pseudomonas aeruginosa, showed that these proteins have modular structures (Fig. ​(Fig.1A).1A). They comprise the α-helical hairpin, the lipoyl-binding domain, and the α-β-barrel domain (2, 9, 14). Mutagenesis and chemical cross-linking studies identified the α-helical hairpin of AcrA as a TolC-binding domain, whereas the α-β-barrel domain was proposed to bind AcrB (6, 11, 12, 16). Surprisingly, in isothermal calorimetry experiments, the core fragment of AcrA without its C-terminal domain (C-domain) was able to bind neither AcrB nor TolC (23). In contrast, the whole-length AcrA interacted with both components. This result suggested that the C-domain of AcrA might be important for these interactions. In crystal structures, however, the C-domains of AcrA and MexA were not resolved, and their structures remain unknown.Open in a separate windowFIG. 1.Proteolytic profiles of AcrA^his in vitro and in vivo. (A) Schematic representation of the secondary structure of AcrA. The unique N-terminal Cys25, which is lipid modified after processing in the periplasm, is shown with an arrow. Positions of amino acid residues that form the α-β-barrel, lipoyl-binding, and α-helical hairpin domains are indicated. AcrA residues cleaved by trypsin are indicated by arrowheads. The 28.9-kDa (K46-R315) core and the 26.5-kDa fragment (K46-R294) are also indicated. (B) Purified AcrA^his (final concentration, 1.95 μM) was digested with trypsin (final concentration, 0.10 μM) at 37°C. Aliquots (10 μl) were taken at different time points, and reactions were terminated by boiling in the SDS sample buffer for 5 min. Tryptic fragments were resolved by SDS-PAGE and analyzed by silver nitrate staining. Minor fragments in the untreated control (0 min) are contaminants that copurify with AcrA^his. Lane M, molecular marker. (C) Proteolytic profiles of AcrA^his in E. coli AG100AX cells carrying pA^his and pA^hisB plasmids. After treatment with increasing concentrations of trypsin for 60 min at 37°C, the whole-cell proteins were resolved by SDS-PAGE and analyzed by immunoblotting with a polyclonal anti-AcrA antibody. Masses of tryptic fragments of the C-domain of AcrA^his identified by mass spectrometry and by mobility in SDS-PAGE are indicated. O.D., optical density as determined by absorbance at 600 nm.The alignment of sequences of highly diverse MFPs from both gram-negative and gram-positive bacteria showed that amino acid sequences of the C-domains are conserved among members of the MFP family (4). In addition, several studies suggested that this region is important for the function of AcrA. The deletion mutant of AcrA lacking 85 C-terminal aa residues is poorly expressed and nonfunctional in multidrug efflux (14). The replacement of aa 290 to 357 of AcrA with an analogous region of YhiU disrupted AcrA function possibly because of the loss of interaction with the AcrB transporter (5). Random mutagenesis of MexA identified C-terminal amino acid residues as important for MexA oligomerization and interaction with MexB (16, 17).In this study, we identified proteolytically labile sites in the C-domain (aa 315 to 397) of the purified AcrA and compared the accessibility of these sites to that in free AcrA and when engaged in the bipartite and tripartite AcrA, AcrB, and TolC interactions in vivo. We found that the assembly of the AcrAB-TolC complex, but not bipartite AcrA-AcrB and AcrA-TolC interactions, protects the C-domain of AcrA from proteolytic digestion. This result suggested that this domain of AcrA interacts with AcrB, TolC, or both. The functional significance of the C-domain was confirmed by site-directed mutagenesis. A single G363C substitution significantly impairs the multidrug efflux activity of AcrAB-TolC.     

Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.     

AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex

Many transporters of Gram-negative bacteria involved in the extracellular secretion of proteins and the efflux of toxic molecules operate by forming intermembrane complexes. These complexes are proposed to span both inner and outer membranes and create a bridge across the periplasm. In this study, we analyzed interactions between the inner and outer membrane components of the tri-partite multidrug efflux pump AcrAB-TolC from Escherichia coli. We found that, once assembled, the intermembrane AcrAB-TolC complex is stable during the separation of the inner and outer membranes and subsequent purification. All three components of the complex co-purify when the affinity tag is attached to either of the proteins suggesting bi-partite interactions between AcrA, AcrB, and TolC. We show that antibiotics, the substrates of AcrAB-TolC, stabilize interactions within the complex. However, the formation of the AcrAB-TolC complex does not require an input of energy.     

Interaction between the TolC and AcrA proteins of a multidrug efflux system of Escherichia coli

This paper provides the biochemical evidence for physical interactions between the outer membrane component, TolC, and the membrane fusion protein component, AcrA, of the major antibiotic efflux pump of Escherichia coli. Cross-linking between TolC and AcrA was independent of the presence of any externally added substrate of the efflux pump or of the pump protein, AcrB. The biochemical demonstration of a TolC-AcrA interaction is consistent with genetic studies in which extragenic suppressors of a mutant TolC strain were found in the acrA gene.     

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

双平板对照筛选跟踪分离细菌外排泵抑制剂

建立了细菌外排泵抑制剂的筛选与活性跟踪方法.准备2个平板,一个为普通营养琼脂平板,另一个为舍小蘖碱的普通营养琼脂平板,通过比较两个平板含药纸片周围抑菌圈的直径大小判断筛选结果,方法可靠稳定.筛选发现某霉菌提取物对细菌外排泵有抑制活性,经活性跟踪分离,得到单体化合物,经NMR鉴定为4′,5,7-三羟基异黄酮.方法简便易行,成本低,适宜于对大批样本进行快速筛选并在分离时进行活性跟踪.     

Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available.     

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.     

Translational Coupling Controls Expression and Function of the DrrAB Drug Efflux Pump

This study investigates the role of translational coupling in the expression and function of DrrA and DrrB proteins, which form an efflux pump for the export of anticancer drugs doxorubicin and daunorubicin in the producer organism Streptomyces peucetius. Interest in studying the role of translational coupling came from the initial observation that DrrA and DrrB proteins confer doxorubicin resistance only when they are expressed in cis. Because of the presence of overlapping stop and start codons in the intergenic region between drrA and drrB, it has been assumed that the translation of drrB is coupled to the translation of the upstream gene drrA even though direct evidence for coupling has been lacking. In this study, we show that the expression of drrB is indeed coupled to translation of drrA. We also show that the introduction of non-coding sequences between the stop codon of drrA and the start of drrB prevents formation of a functional complex, although both proteins are still produced at normal levels, thus suggesting that translational coupling also plays a crucial role in proper assembly. Interestingly, replacement of drrA with an unrelated gene was found to result in very high drrB expression, which becomes severely growth inhibitory. This indicates that an additional mechanism within drrA may optimize expression of drrB. Based on the observations reported here, it is proposed that the production and assembly of DrrA and DrrB are tightly linked. Furthermore, we propose that the key to assembly of the DrrAB complex lies in co-folding of the two proteins, which requires that the genes be maintained in cis in a translationally coupled manner.     

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.     

The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensable for Colicin U Activity but Is Important for Interaction with the Immunity Protein

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575–582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575–582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.     

A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1

Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures–openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores–the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

Targeting Outer Membrane Protein Component AdeC for the Discovery of Efflux Pump Inhibitor against AdeABC Efflux Pump of Multidrug Resistant <Emphasis Type="BoldItalic">Acinetobacter baumannii</Emphasis>

The structure and functioning of multidrug efflux systems provide us with a better understanding of the transport of various antibiotics, thus giving a path for the discovery of effective compounds for combating the multidrug resistance in Acinetobacter baumannii. In the present study, a number of computational techniques have been used to search for an inhibitor for the RND efflux pump, AdeABC, of A. baumannii targeting specifically its outermost component, i.e., AdeC. We have prepared the three-dimensional structure for AdeC using MODELLER v9.16 and identified its active binding site using SiteMap. Using high-throughput virtual screening, we identified compounds from a large library of biogenic compounds on the basis of their effective interaction at the binding site of AdeC. The validation of docking step was performed by plotting ROC curve (enrichment calculations). The docked complexes were further analyzed for their binding free energies by molecular mechanics using Generalized Born model and Solvent Accessibility (MMGBSA). The molecular dynamics simulation was performed for AdeC-ZINC77257599 complex using GROMACS. The present rational drug designing, molecular mechanics and molecular dynamics data provided an inhibitor, i.e, ZINC77257599 [(3R,4Z,6E,8E)-3-hydroxy-2,2,4-trimethyl-10-oxazol-5-yl-deca-4,6,8-trienamide], for the outer membrane protein component (AdeC) of efflux pump AdeABC of A. baumannii.     

Construction of Estimation Regions for the Parameters of Explicit Functional Relationships

Assuming that some explicit functional relationship acts as a mathematical model we consider the case that we are given a finite set of hypercuboids, each of which contains at least one point of the true functional relationship with a certain given probability. We present a procedure to construct sets for the unknown parameters and probabilities for the event that those contain the true parameter value. The procedure is illustrated by an example from pharmaceutical technology.