Application of fragment-based drug discovery to membrane proteins: identification of ligands of the integral membrane enzyme DsbB.
ABSTRACT: Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here, we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The Escherichia coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1071 drug fragments for binding using target immobilized NMR Screening. Biochemical and biophysical validation of the eight most potent hits revealed an IC(50) range of 7-200 microM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins.
Project description:We describe the NMR structure of DsbB, a polytopic helical membrane protein. DsbB, a bacterial cytoplasmic membrane protein, plays a key role in disulfide bond formation. It reoxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. We determined the structure of an interloop disulfide bond form of DsbB, an intermediate in catalysis. Analysis of the structure and interactions with substrates DsbA and quinone reveals functionally relevant changes induced by these substrates. Analysis of the structure, dynamics measurements, and NMR chemical shifts around the interloop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated. Our results demonstrate the extraordinary utility of NMR for functional characterization of polytopic integral membrane proteins and provide insights into the mechanism of DsbB catalysis.
Project description:Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were coexpressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide-bond formation in a range of substrate proteins, in a manner dependent on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way to unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid-bilayer context.
Project description:The integral membrane protein DsbB in Escherichia coli is responsible for oxidizing the periplasmic protein DsbA, which forms disulfide bonds in substrate proteins. We have developed a high-resolution structural model by combining experimental X-ray and solid-state NMR with molecular dynamics (MD) simulations. We embedded the high-resolution DsbB structure, derived from the joint calculation with X-ray reflections and solid-state NMR restraints, into the lipid bilayer and performed MD simulations to provide a mechanistic view of DsbB function in the membrane. Further, we revealed the membrane topology of DsbB by selective proton spin diffusion experiments, which directly probe the correlations of DsbB with water and lipid acyl chains. NMR data also support the model of a flexible periplasmic loop and an interhelical hydrogen bond between Glu26 and Tyr153.
Project description:Disulfide bond formation occurs in secreted proteins in Escherichia coli when the disulfide oxidoreductase DsbA, a soluble periplasmic protein, nonspecifically transfers a disulfide to a substrate protein. The catalytic disulfide of DsbA is regenerated by the inner-membrane protein DsbB. To help identify the specificity determinants in DsbB and to understand the nature of the kinetic barrier preventing direct oxidation of newly secreted proteins by DsbB, we imposed selective pressure to find novel mutations in DsbB that would function to bypass the need for the disulfide carrier DsbA. We found a series of mutations localized to a short horizontal alpha-helix anchored near the outer surface of the inner membrane of DsbB that eliminated the need for DsbA. These mutations changed hydrophobic residues into nonhydrophobic residues. We hypothesize that these mutations may act by decreasing the affinity of this alpha-helix to the membrane. The DsbB mutants were dependent on the disulfide oxidoreductase DsbC, a soluble periplasmic thiol-disulfide isomerase, for complementation. DsbB is not normally able to oxidize DsbC, possibly due to a steric clash that occurs between DsbC and the membrane adjacent to DsbB. DsbC must be in the reduced form to function as an isomerase. In contrast, DsbA must remain oxidized to function as an oxidizing thiol-disulfide oxidoreductase. The lack of interaction that normally exists between DsbB and DsbC appears to provide a means to separate the DsbA-DsbB oxidation pathway and the DsbC-DsbD isomerization pathway. Our mutants in DsbB may act by redirecting oxidant flow to take place through the isomerization pathway.
Project description:The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei A panel of B. pseudomalleidsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.
Project description:In the Escherichia coli system catalysing oxidative protein folding, disulphide bonds are generated by the cooperation of DsbB and ubiquinone and transferred to substrate proteins through DsbA. The structures solved so far for different forms of DsbB lack the Cys104-Cys130 initial-state disulphide that is directly donated to DsbA. Here, we report the 3.4 A crystal structure of a DsbB-Fab complex, in which DsbB has this principal disulphide. Its comparison with the updated structure of the DsbB-DsbA complex as well as with the recently reported NMR structure of a DsbB variant having the rearranged Cys41-Cys130 disulphide illuminated conformational transitions of DsbB induced by the binding and release of DsbA. Mutational studies revealed that the membrane-parallel short alpha-helix of DsbB has a key function in physiological electron flow, presumably by controlling the positioning of the Cys130-containing loop. These findings demonstrate that DsbB has developed the elaborate conformational dynamism to oxidize DsbA for continuous protein disulphide bond formation in the cell.
Project description:Lipid nanodiscs are playing increasingly important roles in studies of the structure and function of membrane proteins. Development of lipid nanodiscs as a membrane-protein-supporting platform, or a drug targeting and delivery vehicle in general, is undermined by the fluidic and labile nature of lipid bilayers. Here, we report the discovery of polymer nanodiscs, i.e., discoidal amphiphilic block copolymer membrane patches encased within membrane scaffold proteins, as a novel two-dimensional nanomembrane that maintains the advantages of lipid nanodiscs while addressing their weaknesses. Using MsbA, a bacterial ATP-binding cassette transporter as a membrane protein prototype, we show that the protein can be reconstituted into the polymer nanodiscs in an active state. As with lipid nanodiscs, reconstitution of detergent-solubilized MsbA into the polymer nanodiscs significantly enhances its activity. In contrast to lipid nanodiscs that undergo time- and temperature-dependent structural changes, the polymer nanodiscs experience negligible structural evolution under similar environmental stresses, revealing a critically important property for the development of nanodisc-based characterization methodologies or biotechnologies. We expect that the higher mechanical and chemical stability of block copolymer membranes and their chemical versatility for adaptation will open new opportunities for applications built upon diverse membrane protein functions, or involved with drug targeting and delivery.
Project description:Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB, and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ~500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge-transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (~20 ppm), methoxys (~60 ppm), olefin carbons (120-140 ppm), and carbonyls (150-160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge-transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.
Project description:Nanodiscs constitute a tool for the solubilization of membrane proteins in a lipid bilayer, thus offering a near-native membrane environment. Many membrane proteins interact with other membrane proteins; however, the co-reconstitution of multiple membrane proteins in a single nanodisc is a random process that is adversely affected by several factors, including protein aggregation. Here, we present an approach for the controlled co-reconstitution of multiple membrane proteins in a single nanodisc. The temporary attachment of designated oligonucleotides to individual membrane proteins enables the formation of stable, detergent-solubilized membrane protein complexes by base-pairing of complementary oligonucleotide sequences, thus facilitating the insertion of the membrane protein complex into nanodiscs with defined stoichiometry and composition. As a proof of principle, nanodiscs containing a heterodimeric and heterotrimeric membrane protein complex were reconstituted using a fluorescently labeled voltage-gated anion channel (VDAC) as a model system.
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.