Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD.

Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10-6 and 10-9 M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 104 and 106 M-1s-1. For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.

nDsbD: a redox interaction hub in the Escherichia coli periplasm.

DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a 'redox hub' with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners.

Protein-mediated boundary lubrication in arthroplasty.

Wear of articulated surfaces can be a major lifetime-limiting factor in arthroplasty. In the natural joint, lubrication is effected by the body's natural synovial fluid. Following arthroplasty, and the subsequent reformation of the synovial membrane, a fluid of similar composition surrounds the artificial joint. Synovial fluid contains, among many other constituents, a substantial concentration of the readily adsorbing protein albumin. The ability of human serum albumin to act as a boundary lubricant in joint prostheses has been investigated using a pin-on-disc tribometer. Circular dichroism spectroscopy was employed to follow the temperature- and time-dependent conformational changes of human serum albumin in the model lubricant solution. Effects of protein conformation and polymer surface hydrophilicity on protein adsorption and the resulting friction in the boundary lubrication regime have been investigated. Unfolded proteins preferentially adsorb onto hydrophobic polymer surfaces, where they form a compact, passivating layer and increase sliding friction-an effect that can be largely suppressed by rendering the substrate more hydrophilic. A molecular model for protein-mediated boundary friction is proposed to consolidate the observations. The relevance of the results for in vivo performance and ex vivo hip-joint testing are discussed.

Uroplakin Ia is the urothelial receptor for uropathogenic Escherichia coli: evidence from in vitro FimH binding.

The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment.

Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.

Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA.

The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.

Characterization of FimC, a periplasmic assembly factor for biogenesis of type 1 pili in Escherichia coli.

Assembly of type 1 pili from Escherichia coli is mediated by FimC, a periplasmic chaperone (assembly factor) consisting of two immunoglobulin-like domains. FimC is assumed to recognize the individual pilus subunits in the periplasm mainly via their conserved C-terminal segments and to deliver the subunits to an assembly platform in the outer membrane. Here we present the first biochemical characterization of a periplasmic pilus chaperone and analyze the importance of the two chaperone domains for stability and function. Comparison of the isolated C-terminal domain with wild-type FimC revealed a strongly reduced thermodynamic stability, indicating strong interdomain interactions. The affinity of FimC toward a peptide corresponding to the 11 C-terminal residues of the type 1 pilus adhesin FimH is at least 1000-fold lower compared to binding of intact FimH, confirming that bacterial pilus chaperones, unlike other chaperones, specifically interact with folded pilus subunits.

Influence of the pK(a) value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases.

Thioredoxin constitutes the prototype of the thiol-disulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the consensus sequence Cys-Xaa-Xaa-Cys. The more N-terminal active-site cysteine is generally a strong nucleophile with an abnormal low pK(a) value. In contrast, the more C-terminal cysteine is buried and only little is known about its effective pK(a) during catalysis of disulfide exchange reactions. Here we have analyzed the pK(a) values of the active-site thiols in wild type thioredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively (13)C(beta)-Cys-labeled proteins. We find that the effective pK(a) of the buried cysteine (pK(b)) of the variant is increased, while the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relative to the corresponding pK(a) values in the wild type. We propose two empirical models which exclusively require the knowledge of pK(N) to predict the redox properties of thiol-disulfide oxidoreductases with reasonable accuracy.

Fluorescence quenching in the DsbA protein from Escherichia coli: complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan.

The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and is required for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76 and W126. The fluorescence of W76 changes upon reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391-6400). The fluorescence of W126 is highly quenched. The only two potential side chain quenchers are Q74 and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity. The fluorescence intensity increase is not due to the removal of dynamic quenchers but to an increase in the population with the longest lifetime. In this report, the possibility of a change in the conformation of W126 is investigated theoretically by using molecular mechanics and dynamic simulations and experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and the experimental results reveal that the amino acid replacements allow W126 to increase the population of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors' knowledge, this is the first time that the kinetics of Trp population reshuffling have been measured.

Circularly permuted variants of the green fluorescent protein.

Folding of the green fluorescent protein (GFP) from Aequorea victoria is characterized by autocatalytic formation of its p-hydroxybenzylideneimidazolidone chromophore, which is located in the center of an 11-stranded beta-barrel. We have analyzed the in vivo folding of 20 circularly permuted variants of GFP and find a relatively low tolerance towards disruption of the polypeptide chain by introduction of new termini. All permuted variants with termini in strands of the beta-barrel and about half of the variants with termini in loops lost the ability to form the chromophore. The thermal stability of the permuted GFPs with intact chromophore is very similar to that of the wild-type, indicating that chromophore-side chain interactions strongly contribute to the extraordinary stability of GFP.

Importance of redox potential for the in vivo function of the cytoplasmic disulfide reductant thioredoxin from Escherichia coli.

The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.

Peptides and proteins in neurodegenerative disease: helix propensity of a polypeptide containing helix 1 of the mouse prion protein studied by NMR and CD spectroscopy.

Transmissible spongiform encephalopathies (TSE) or "prion diseases" have been related to the "protein-only hypothesis", which suggests that the "scrapie form (PrPSc)" of the prion protein (PrP) is the TSE infectious agent. The nmr structure of the ubiquitous benign cellular form of PrP (PrPC) consists of a globular domain of residues 126-231 with three alpha-helices and a short beta-sheet, and a flexible extended "tail" of residues 23-125. The peptide segment of helix 1 has been implicated in various stages of hypothetical pathways to prion pathology on the basis of the protein-only idea, including that it takes part in the conformation change that leads from PrPC to PrPSc. In this paper we describe solution nmr and circular dichroism studies of the synthetic hexadecapeptide mPrP(143-158), with the sequence H-NDWEDRYYRENMYRYP-NH2, where the bold letters represent the segment that forms helix 1 in murine PrPC. In both H2O and a 1:1 mixture of H2O and trifluoroethanol this polypeptide segment shows high helix propensity, which is a key issue in discussions on potential roles of this molecular region in conformational transitions of PrP.

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.

Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates.

The kinetics of folding of mPrP(121-231), the structured 111-residue domain of the murine cellular prion protein PrP(C), were investigated by stopped-flow fluorescence using the variant F175W, which has the same overall structure and stability as wild-type mPrP(121-231) but shows a strong fluorescence change upon unfolding. At 22 degrees C and pH 7.0, folding of mPrP(121-231)-F175W is too fast to be observable by stopped-flow techniques. Folding at 4 degrees C occurs with a deduced half-life of approximately 170 micros without detectable intermediates, possibly the fastest protein-folding reaction known so far. Thus, propagation of the abnormal, oligomeric prion protein PrP(Sc), which is supposed to be the causative agent of transmissible spongiform encephalopathies, is unlikely to follow a mechanism where kinetic folding intermediates of PrP(C) are a source of PrP(Sc) subunits.

Pilus chaperone FimC-adhesin FimH interactions mapped by TROSY-NMR.

The 23 kDa two-domain periplasmic chaperone FimC from Escherichia coli is required for the assembly of type-1 pili, which are filamentous, highly oligomeric protein complexes anchored to the outer bacterial membrane that mediate adhesion of pathogenic E. coli strains to host cell surfaces. Here we identified the contact sites on the surface of the NMR structure of FimC that are responsible for the binding of the 28 kDa mannose-binding type-1 pilus subunit FimH by 15N and 1H NMR chemical shift mapping, using transverse relaxation-optimized spectroscopy (TROSY). The FimH-binding surface of FimC is formed nearly entirely by the N-terminal domain, and its extent and shape indicate that FimC binds a folded form of the pilus subunits.

Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein.

Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121-231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121-231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121-231) and similar to full-length PrP(23-231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation. The thermodynamic stabilities of the variants do not correlate with specific disease phenotypes. Five of the amino acid replacements destabilize PrP(121-231), but the other variants have the same stability as the wild-type protein. These data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.

Random circular permutation of DsbA reveals segments that are essential for protein folding and stability.

One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state. In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo. A total of 51 different active variants were identified. The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures. New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities. In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type. We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability.

Conversion of a catalytic into a structural disulfide bond by circular permutation.

The thiol-disulfide oxidoreductase DsbA from Escherichia coli is the strongest oxidant of the enzyme family and required for disulfide bond formation in the bacterial periplasm. The catalytic domain of this 189-residue protein has a thioredoxin-like fold and contains a catalytic disulfide bridge that is located within the sequence Cys30-Pro31-His32-Cys33 at the N-terminus of an alpha-helix. The Cys30-Cys33 disulfide bond destabilizes DsbA by about 16 kJ/mol at pH 7.0, which appears to be caused by the extremely low pKa value of approximately 3.4 of the nucleophilic Cys30 thiol. Here we report the characterization of a circularly permuted variant of DsbA, termed H32-P31, in which the natural termini are connected by a Gly3-Thr-Gly linker and the new termini are located between the active-site cysteines (first residue His32, last residue Pro31). The disulfide bond in the variant thus connects the second with the penultimate residue. H32-P31 adopts a wild-type-like structure and folds reversibly and cooperatively in both redox forms. However, the permuted variant is catalytically inactive as dithiol oxidase in vivo and in vitro. Both cysteine thiols have pKa values > 8; the variant is 500-fold more reducing than the wild type and more stable in its oxidized form. Thus, the Cys30-Cys33 disulfide in the variant H32-P31 has adopted properties of a structural disulfide bond.