Biomedical subjects

Lode Wyns

Publications and source records attributed to Lode Wyns.

At least 19 recordsLinked to original sources

PubMed2005

Molecular basis of gyrase poisoning by the addiction toxin CcdB.

Gyrase is an ubiquitous bacterial enzyme that is responsible for disentangling DNA during DNA replication and transcription. It is the target of the toxin CcdB, a paradigm for plasmid addiction systems and related bacterial toxin-antitoxin systems. The crystal structure of CcdB and the dimerization domain of the A subunit of gyrase (GyrA14) dictates an open conformation for the catalytic domain of gyrase when CcdB is bound. The action of CcdB is one of a wedge that stabilizes a dead-end covalent gyrase:DNA adduct. Although CcdB and GyrA14 form a globally symmetric complex where the two 2-fold axes of both dimers align, the complex is asymmetric in its details. At the centre of the interaction site, the Trp99 pair of CcdB stacks with the Arg462 pair of GyrA14, explaining why the Arg462Cys mutation in the A subunit of gyrase confers resistance to CcdB. Overexpression of GyrA14 protects Escherichia coli cells against CcdB, mimicking the action of the antidote CcdA.

Bacterial Proteins↗

PubMed2005

Encapsulation of therapeutic nucleoside hydrolase in functionalised nanocapsules.

Liposomes are introduced as encapsulating carrier for prodrug activating enzymes. Inosineã-adenosineã-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in porin functionalized dioleyl-phosphatidylglycerol/egg-phosphatidylglycerol (DOPC/EPG) liposomes. Reactors had radiuses in the nanometer scale. First, transport of nucleosides through general diffusion porins OmpF and PhoE was measured in swelling assays, after which fully functional nanoreactors were developed. Enzyme catalysis of p-nitrophenylriboside, a substrate analogue for nucleoside hydrolases, was significantly higher in permeabilized vesicles than in control vesicles without porins. Residual activity of control vesicles possibly resides in an interaction between the enzyme and the liposomes. This interaction was not of electrostatic nature, since it remained unaffected after the addition of high salt or after perturbation of liposome surface charge and charge density. With these vesicles, we have introduced a new strategy for prodrug therapy, combining the benefits of ADEPT and liposome targeting strategies.

Drug Compounding↗

PubMed2005

Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires.

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.

Amino Acid Sequence↗

PubMed2005

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin.

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha-D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd = 0.15 microM) than mannose (Kd = 2.3 microM). Exploration of the binding affinities of alpha- d-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Adhesins, Escherichia coli↗

PubMed2004

Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations.

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining alpha-helices that form the core of the alpha-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, and the associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils.

Amino Acid Substitution↗

PubMed2004

Overexpression, purification and crystallization of bacteriocin LlpA from Pseudomonas sp. BW11M1.

LlpA is a bacteriocin produced by Pseudomonas sp. BW11M1 that shows remarkable similarity to a family of mannose-binding plant lectins. A His-tagged version of LlpA was recombinantly produced in Escherichia coli and purified to homogeneity. Single crystals were grown by vapour diffusion and belong to space group P2(1)2(1)2, with unit-cell parameters a = 150.5, b = 154.5, c = 34.2 A. The crystals diffract to at least 2.2 A using synchrotron radiation.

Bacteriocins↗

PubMed2004

Chemical basis for the affinity maturation of a camel single domain antibody.

Affinity maturation of classic antibodies supposedly proceeds through the pre-organization of the reactive germ line conformational isomer. It is less evident to foresee how this can be accomplished by camelid heavy-chain antibodies lacking light chains. Although these antibodies are subjected to somatic hypermutation, their antigen-binding fragment consists of a single domain with restricted flexibility in favor of binding energy. An antigen-binding domain derived from a dromedary heavy-chain antibody, cAb-Lys3, accumulated five amino acid substitutions in CDR1 and CDR2 upon maturation against lysozyme. Three of these residues have hydrophobic side chains, replacing serines, and participate in the hydrophobic core of the CDR1 in the mature antibody, suggesting that conformational rearrangements might occur in this loop during maturation. However, transition state analysis of the binding kinetics of mature cAb-Lys3 and germ line variants show that the maturation of this antibody relies on events late in the reaction pathway. This is reflected by a limited perturbation of k(a) and a significantly decreased k(d) upon maturation. In addition, binding reactions and the maturation event are predominantly enthalpically driven. Therefore, maturation proceeds through the increase of favorable binding interactions, or by the reduction of the enthalpic penalty for desolvation, as opposed to large entropic penalties associated with conformational changes and structural plasticity. Furthermore, the crystal structure of the mutant with a restored germ line CDR2 sequence illustrates that the matured hydrophobic core of CDR1 in cAb-Lys3 might be compensated in the germ line precursor by burying solvent molecules engaged in a stable hydrogen-bonding network with CDR1 and CDR2.

Amino Acid Sequence↗

PubMed2004

SABmark--a benchmark for sequence alignment that covers the entire known fold space.

The Sequence Alignment Benchmark (SABmark) provides sets of multiple alignment problems derived from the SCOP classification. These sets, Twilight Zone and Superfamilies, both cover the entire known fold space using sequences with very low to low, and low to intermediate similarity, respectively. In addition, each set has an alternate version in which unalignable but apparently similar sequences are added to each problem.

Algorithms↗

PubMed2004

How thioredoxin can reduce a buried disulphide bond.

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10-Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10-Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10-Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol-disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.

Disulfides↗

PubMed2004

The structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adduct.

Structural insights into formation of the complex between the ubiquitous thiol-disulfide oxidoreductase thioredoxin and its oxidized substrate are under-documented owing to its entropical instability. In vitro, it is possible via a reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) to make a stable mixed-disulfide complex between thioredoxin from Staphylococcus aureus and one of its substrates, oxidized pI258 arsenate reductase (ArsC) from S. aureus. In the absence of the crystal structure of an ArsC-thioredoxin complex, the structures of two precursors of the complex, the ArsC triple mutant ArsC C10SC15AC82S and its 5-thio-2-nitrobenzoic acid (TNB) adduct, were determined. The ArsC triple mutant has a structure very similar to that of the reduced form of wild-type ArsC, with a folded redox helix and a buried catalytic Cys89. In the adduct form, the TNB molecule is buried in a hydrophobic pocket and the disulfide bridge between TNB and Cys89 is sterically inaccessible to thioredoxin. In order to form a mixed disulfide between ArsC and thioredoxin, a change in the orientation of the TNB-Cys89 disulfide in the structure is necessary.

Arsenite Transporting ATPases↗

PubMed2004

Crystallization of CcdB in complex with a GyrA fragment.

Plasmid addiction systems consist of a plasmid-encoded toxin-antidote pair that serves to stabilize low-copy-number plasmids in bacterial populations. CcdB, the toxin from the ccd system on the Escherichia coli F plasmid, acts as a gyrase poison. A 14 kDa fragment of gyrase, GyrA14, was found to bind to the toxin CcdB with an affinity of 1.75 x 10(-8) M. Crystals of the (GyrA14)(2) dimer in its free state belong to space group P4(3)2(1)2, with unit-cell parameters a = 86.4, c = 89.4 angstroms, and diffract to 2.4 angstroms. Crystals of the (GyrA14)(2)-(CcdB)(2) complex belong to space group P2(1)2(1)2(1), with a = 52.1, b = 83.3, c = 110.9 angstroms, and diffract to 2.8 angstroms resolution.

Bacterial Proteins↗

PubMed2004

Untangle, a tool for filtering overlapping diffraction patterns from multicrystals.

Standard crystallographic data-processing protocols are based on single-crystal models; data from aggregates of multiple crystals with different orientations are difficult to process. In certain cases, it is possible to separately index the diffraction patterns from the dominant crystals in the aggregate. Untangle is a program designed to identify and eliminate overlapping spots from such patterns in order to improve data quality. The program has a Python core with a simple and highly portable graphical user interface, permitting visual verification of the process and interactive modification of the overlap threshold. The software is available under an open-source license.

Bacterial Proteins↗

PubMed2004

Align-m--a new algorithm for multiple alignment of highly divergent sequences.

MOTIVATION: Multiple alignment of highly divergent sequences is a challenging problem for which available programs tend to show poor performance. Generally, this is due to a scoring function that does not describe biological reality accurately enough or a heuristic that cannot explore solution space efficiently enough. In this respect, we present a new program, Align-m, that uses a non-progressive local approach to guide a global alignment. RESULTS: Two large test sets were used that represent the entire SCOP classification and cover sequence similarities between 0 and 50% identity. Performance was compared with the publicly available algorithms ClustalW, T-Coffee and DiAlign. In general, Align-m has comparable or slightly higher accuracy in terms of correctly aligned residues, especially for distantly related sequences. Importantly, it aligns much fewer residues incorrectly, with average differences of over 15% compared with some of the other algorithms. AVAILABILITY: Align-m and the test sets are available at http://bioinformatics.vub.ac.be

Algorithms↗

PubMed2004

Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin.

The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.

Amino Acid Sequence↗

PubMed2003

Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm.

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::beta-lactamase fusion activating a prodrug.

Animals↗

PubMed2003

Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa.

The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose. All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains. The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose. In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential. In the fucose complex, no such defects are observed. Instead, C6 makes favourable interactions with a small hydrophobic patch. Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.

Binding Sites↗

PubMed2003

A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme.

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues, often with serious pathological consequences. Non-neuropathic systemic amyloidosis is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H. Our structural studies reveal that the epitope includes neither the site of mutation nor most residues in the region of the protein structure that is destabilized by the mutation. Instead, the binding of the antibody fragment achieves its effect by restoring the structural cooperativity characteristic of the wild-type protein. This appears to occur at least in part through the transmission of long-range conformational effects to the interface between the two structural domains of the protein. Thus, reducing the ability of an amyloidogenic protein to form partly unfolded species can be an effective method of preventing its aggregation, suggesting approaches to the rational design of therapeutic agents directed against protein deposition diseases.

Amyloidosis↗

PubMed2003

Purification of an oxidation-sensitive enzyme, pI258 arsenate reductase from Staphylococcus aureus.

Arsenate reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (K(a)=1.1 mM) and potassium was stabilizing ArsC (DeltaT(m)=+6.2 degrees C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture.

Arsenite Transporting ATPases↗