Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter.
ABSTRACT: Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here, we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic-facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.
Project description:In BtuB, the Escherichia coli TonB-dependent transporter for vitamin B12, substrate binding to the extracellular surface unfolds a conserved energy coupling motif termed the Ton box into the periplasm. This transmembrane signaling event facilitates an interaction between BtuB and the inner-membrane protein TonB. In this study, continuous-wave and pulse electron paramagnetic resonance in a native outer-membrane preparation demonstrate that signaling also occurs from the periplasmic to the extracellular surface in BtuB. The binding of a TonB fragment to the periplasmic interface alters the configuration of the second extracellular loop and partially dissociates a spin-labeled substrate analog. Moreover, mutants in the periplasmic Ton box that are transport-defective alter the binding site for vitamin B12 in BtuB. This work demonstrates that the Ton box and the extracellular substrate binding site are allosterically coupled in BtuB, and that TonB binding may initiate a partial round of transport.
Project description:An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron-electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment.
Project description:The α-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb) proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, α-DsbA1 and α-DsbA2, and an integral membrane protein, α-DsbB. α-DsbA1 and α-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein α-DsbB oxidizes α-DsbA1, but not α-DsbA2. The interaction between α-DsbA1 and α-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of α-DsbB. In the electron flow cascade, oxidation of α-DsbA1 by α-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with α-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in α-DsbA1, conserved in other α-proteobacterial DsbAs but not in γ-proteobacterial DsbAs. This is the first characterization of the integral membrane protein α-DsbB from Wolbachia and reveals that the non-catalytic cysteines of α-DsbA1 regulate the redox relay system in cooperation with α-DsbB.
Project description:BtuB is a large outer-membrane ?-barrel protein that belongs to a class of active transport proteins that are TonB-dependent. These TonB-dependent transporters are based upon a 22-stranded antiparallel ?-barrel, which is notably asymmetric in its length. Here, site-directed spin labeling and simulated annealing were used to locate the membrane lipid interface surrounding BtuB when reconstituted into phosphatidylcholine bilayers. Positions on the outer facing surface of the ?-barrel and the periplasmic turns were spin-labeled and distances from the label to the membrane interface estimated by progressive power saturation of the electron paramagnetic resonance spectra. These distances were then used as atom-to-plane distance restraints in a simulated annealing routine, to dock the protein to two independent planes and produce a model representing the average position of the lipid phosphorus atoms at each interface. The model is in good agreement with the experimental data; however, BtuB is mismatched to the bilayer thickness and the resulting planes representing the bilayer interface are not parallel. In the model, the membrane thickness varies by 11 Å around the circumference of the protein, indicating that BtuB distorts the bilayer interface so that it is thinnest on the short side of the protein ?-barrel.
Project description:Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg(E) from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg(E) domain was created using the homodimeric, four-helix bundle structure of the Tar(S) domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg(E) periplasmic domain is very similar to the Tar(S) domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.
Project description:Intimin is a bacterial adhesin located on the surface of enteropathogenic Escherichia coli and other related bacteria that is believed to self-translocate across the outer membrane (OM), and therefore it has been regarded as a member of the type V secretion system (T5SS), which includes classical autotransporters (ATs). However, intimin has few structural similarities to classical ATs and an opposite topology with an OM-embedded N region and a secreted C region. Since the actual secretion mechanism of intimin is unknown, we investigated intimin biogenesis by analyzing its requirement of periplasmic chaperones (DsbA, SurA, Skp, and DegP) and of OM protein BamA (YaeT/Omp85) for folding, OM insertion, and translocation. Using full-length and truncated intimin polypeptides, we demonstrate that DsbA catalyzes the formation of a disulfide bond in the D3 lectin-like domain of intimin in the periplasm, indicating that this secreted C-terminal domain is at least partially folded prior to its translocation across the OM. We also show that SurA chaperone plays the major role for periplasmic transport and folding of the N region of intimin, whereas the parallel pathway made by Skp and DegP chaperones plays a secondary role in this process. Further, we demonstrate that BamA is essential for the insertion of the N region of intimin in the OM and that the protease activity of DegP participates in the degradation of misfolded intimin. The significance of these findings for a BamA-dependent secretion mechanism of intimin is discussed in the context of T5SSs.
Project description:Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.
Project description:Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.
Project description:A disulfide-bond formation system for nascent proteins in the Escherichia coli periplasm contains efficient electron transfer systems for the catalysis of oxidation. This electrochemical system has interesting implications in vivo. Disulfide bonds are formed by disulfide-bond formation protein A (DsbA), which contains two reactive cysteines. DsbA is reoxidized by a membrane protein, disulfide-bond formation protein B (DsbB), which has four catalytic cysteines. The oxidation of DsbA by DsbB seems energetically unfavorable on the basis of the redox potential. The oxidizing power of ubiquinone (UQ), which endogenously binds with DsbB, is believed to promote this reaction. However, using UQ-deficient DsbB, it was found that the oxidation of DsbA by DsbB proceeds independently of UQ. Thus, the reaction mechanism of DsbA oxidation by DsbB is under debate. In this study, we used the quartz crystal microbalance technique, which detects the intermediate complex between DsbA and DsbB during DsbA oxidation as a change in mass, to obtain kinetic parameters of DsbA oxidation under both the oxidized and reduced states of UQ at acidic and basic pH. In addition, we utilized sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility shift assay technique to determine the pK a of the cysteine thiol groups in DsbA and DsbB. We found that DsbA oxidation proceeded independently of UQ and was greatly affected in kinetics by the shuffling of electrons among the four cysteine residues in DsbB, regardless of pH. These results suggest that DsbA oxidation is driven in an entropy-dependent manner, in which the electron-delocalized intermediate complex is stabilized by preventing a reverse reaction. These findings could contribute to the design of bio-inspired electrochemical systems for industrial applications.
Project description:As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.