Biomedical subjects

Lieven Buts

Publications and source records attributed to Lieven Buts.

8 recordsLinked to original sources

PubMed2005

Impact of natural variation in bacterial F17G adhesins on crystallization behaviour.

Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies.

Adhesins, Bacterial↗

PubMed2005

Crystallization and crystal manipulation of the Pterocarpus angolensis seed lectin.

The Man/Glc-specific legume lectin from the seeds of the African bloodwood tree (Pterocarpus angolensis) was crystallized in the presence of the disaccharide ligand Man(alpha1-3)ManMe. Small crystals initially appeared from a preliminary screen, but proved difficult to reproduce. The initial crystals were used to prepare microseeds, leading to a reproducible crystallization protocol. All attempts to obtain crystals directly of the ligand-free protein or of other carbohydrate complexes failed. However, the Man(alpha1-3)ManMe co-crystals withstand soaking with ten other carbohydrates known to bind to the lectin. Soaking for 15 min in 100 mM carbohydrate typically resulted in complete replacement of Man(alpha1-3)ManMe by the desired carbohydrate despite the involvement of lattice contacts at the binding site. Transferring the crystals for two weeks in carbohydrate-free artificial mother liquor resulted in the complete removal of the sugar from one of the two monomers in the asymmetric unit. Additional treatment of these crystals with 100 mM EDTA for two weeks resulted in removal of the structural calcium and manganese ions, which is accompanied by significant structural rearrangements of the loops that constitute the carbohydrate-binding site.

Crystallography, X-Ray↗

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↗

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↗

PubMed2003

Solving the phase problem for carbohydrate-binding proteins using selenium derivatives of their ligands: a case study involving the bacterial F17-G adhesin.

The Escherichia coli adhesin F17-G is a carbohydrate-binding protein that allows the bacterium to attach to the intestinal epithelium of young ruminants. The structure of the 17 kDa lectin domain of F17-G was determined using the anomalous dispersion signal of a selenium-containing analogue of the monosaccharide ligand N-acetyl-d-glucosamine in which the anomeric oxygen was replaced by an Se atom. A three-wavelength MAD data set yielded good experimental phases to 2.6 A resolution. The structure was refined to 1.75 A resolution and was used to solve the structures of the ligand-free protein and the F17-G-N-acetyl-d-glucosamine complex. This selenium-carbohydrate phasing method could be of general use for determining the structures of carbohydrate-binding proteins.

Acetylglucosamine↗

PubMed2003

Crystal structure of Pterocarpus angolensis lectin in complex with glucose, sucrose, and turanose.

The crystal structure of the Man/Glc-specific seed lectin from Pterocarpus angolensis was determined in complex with methyl-alpha-d-glucose, sucrose, and turanose. The carbohydrate binding site contains a classic Man/Glc type specificity loop. Its metal binding loop on the other hand is of the long type, different from what is observed in other Man/Glc-specific legume lectins. Glucose binding in the primary binding site is reminiscent of the glucose complexes of concanavalin A and lentil lectin. Sucrose is found to be bound in a conformation similar as seen in the binding site of lentil lectin. A direct hydrogen bond between Ser-137(OG) to Fru(O2) in Pterocarpus angolensis lectin replaces a water-mediated interaction in the equivalent complex of lentil lectin. In the turanose complex, the binding site of the first molecule in the asymmetric unit contains the alphaGlc1-3betaFruf form of furanose while the second molecule contains the alphaGlc1-3betaFrup form in its binding site.

Binding Sites↗

PubMed2003

The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine.

The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-beta-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 A resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM-1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal beta-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion.

Acetylglucosamine↗