Biomedical subjects

Bart Hazes

Publications and source records attributed to Bart Hazes.

15 recordsLinked to original sources

PubMed2006

Purification, kinetic characterization, and mapping of the minimal catalytic domain and the key polar groups of Helicobacter pylori alpha-(1,3/1,4)-fucosyltransferases.

The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.

Catalytic Domain↗

PubMed2005

A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori {alpha}1,3/4 fucosyltransferase determines substrate specificity.

Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893-21900) demonstrated that exchange of the hypervariable loops, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.

Amino Acid Sequence↗

PubMed2005

A modified vapor-diffusion crystallization protocol that uses a common dehydrating agent.

In the vapor-diffusion protein-crystallization method, a small drop containing protein sample mixed with a crystallization solution is equilibrated against a reservoir solution in a sealed chamber. Whereas the chemical composition of the crystallization solution is critical for success, the primary role of the reservoir solution is to slowly concentrate the crystallization drop in a controlled fashion. Accordingly, it might be possible to use any reservoir solution of appropriate dehydrating strength. The important practical consequence is that many different experiments can share the same reservoir solution. This approach, called the ;shared reservoir solution' method, significantly simplifies manual and robotic experiment setup, reduces cost and allows a completely new design of optically superior and higher density crystallization plates. Although this research was motivated by these practical advantages, recent reports and the authors' results indicate that this method may actually increase crystallization success. The authors suggest that this may indicate that a protein has a preferred water activity for crystallization. Here, present practical and theoretical considerations as well as experimental tests of the shared reservoir solution method are presented.

Aldose-Ketose Isomerases↗

PubMed2005

A nanovolume crystallization robot that creates its crystallization screens on-the-fly.

Protein crystallization generally consists of an initial screen followed by optimization of promising conditions. Whereas the initial screen typically uses a standard set of pre-made crystallization cocktails, optimization requires new cocktails with small perturbations of the original composition. Highly parallel synchronous crystallization robots are ideal for initial screening, but they depend on pre-made crystallization cocktails. Asynchronous crystallization robots can create crystallization cocktails from stock solutions, but in practice this ability is rarely exploited. Instead, large-scale operations typically use a general liquid-handling robot to create optimization screens, whereas academics mostly rely on manual optimization. Here, the use of an asynchronous crystallization robot to create customized crystallization cocktails and set up nanovolume crystallization experiments without a compromise in speed or drop quality is described. This approach avoids the complex integration of hardware, software and dataflow between two robots and saves cost and space. As a proof of principle, a commercial crystal screen has been reproduced with the robot and shows that results are virtually identical to using the actual commercial screen.

Combinatorial Chemistry Techniques↗

PubMed2005

Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin.

Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized. EVM053 crystallizes in space group C222(1), with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 A. Diffraction data have been processed to 1.8 A resolution and a self-rotation function indicates that there are two molecules per asymmetric unit.

Cloning, Molecular↗

PubMed2005

Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand.

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the beta4-beta5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49G(B6) and a Ly-49G(B6)-like receptor, Ly-49M(nonobese diabetic), but not Ly-49G(BALB/c). By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the beta4-beta5 loop and the beta1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49G(B6) ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the beta4-beta5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

Amino Acid Sequence↗

PubMed2005

F-like type IV secretion systems encode proteins with thioredoxin folds that are putative DsbC homologues.

F and R27 are conjugative plasmids of enteric bacteria belonging to the IncF and IncHI1 plasmid incompatibility groups, respectively. Based on sequence analysis, two genes of the F transfer region, traF and trbB, and three genes of the R27 transfer region, trhF, dsbC, and htdT, are predicted to encode periplasmic proteins containing a C-terminal thioredoxin fold. The C-X-X-C active-site motif of thioredoxins is present in all of these proteins except TraF(F). Escherichia coli carrying a dsbA mutation, which is deficient in disulfide bond formation, cannot synthesize pili and exhibits hypersensitivity to dithiothreitol (DTT) as monitored by mating ability. Overproduction of the E. coli disulfide bond isomerase DsbC, TrbB(F), DsbC(R27), or HtdT(R27), but not TraF(F) or TrhF(R27), reverses this hypersensitivity to DTT. Site-directed mutagenesis established that the C-X-X-C motif was necessary for this activity. Secretion into the periplasm of the C-terminal regions of TrbB(F) and DsbC(R27), containing putative thioredoxin folds, but not TrhF(R27), partially complemented the host dsbA mutation. A trbB(F) deletion mutant showed a 10-fold-lower mating efficiency in an E. coli dsbC null strain but had no phenotype in wild-type E. coli, suggesting redundancy in function between TrbB(F) and E. coli DsbC. Our results indicate that TrbB(F), DsbC(R27), and HtdT(R27) are putative disulfide bond isomerases for their respective transfer systems. TraF(F) is essential for conjugation but appears to have a function other than disulfide bond chemistry.

Amino Acid Motifs↗

PubMed2004

Pros and cons of cryocrystallography: should we also collect a room-temperature data set?

High-resolution protein structures are becoming more common owing to the availability of increasingly brilliant synchrotron X-ray sources. However, to withstand the increased X-ray dose the crystals must be held at cryogenic temperatures. To compare the benefit of increased resolution with the drawback of potential temperature-induced changes, three room-temperature and three cryogenic data sets for PAK pilin have been collected at resolutions between 1.8 and 0.78 A. The results show that although the high-resolution cryogenic structures are more precise and more detailed, they also show systematic deviations from the room-temperature structures. Small but significant differences are even observed in the structural core, whilst more extensive changes occur at the protein surface. These differences can affect biological interpretations, especially because many important biological processes take place at the protein surface. Accordingly, although high-quality cryogenic synchrotron data is extremely valuable to protein crystallography, room-temperature structures are still desirable, especially if the research question involves protein features that are sensitive to temperature-induced changes.

Crystallography↗

PubMed2004

Crystallization and preliminary diffraction studies of TraF, a component of the Escherichia coli type IV secretory system.

TraF, a component of the Escherichia coli type IV secretory system, has been crystallized and preliminary X-ray diffraction data have been collected. TraF is a 26 kDa protein encoded by the E. coli F plasmid and is required for conjugative plasmid transfer and the formation of sex pili. The N-terminal domain of TraF has no recognizable sequence features, whereas the C-terminal domain is believed to adopt a thioredoxin fold. However, since the active-site cysteines of thioredoxin-like proteins are not conserved in TraF, its biochemical role remains unclear. TraF crystallizes in space group C2, with unit-cell parameters a = 119.87, b = 34.36, c = 46.21 A, beta = 90.40 degrees , and crystals diffract to 2.3 A resolution.

Crystallization↗

PubMed2004

Crystallographic analysis of the Pseudomonas aeruginosa strain K122-4 monomeric pilin reveals a conserved receptor-binding architecture.

Adherence of pathogens to host cells is critical for the initiation of infection and is thus an attractive target for anti-infective therapeutics and vaccines. In the opportunistic human pathogen Pseudomonas aeruginosa, host-cell adherence is achieved predominantly by type IV pili. Analysis of several clinical strains of P. aeruginosa reveals poor sequence conservation between pilin genes, including the residues in the receptor-binding site. Interestingly, the receptor-binding sites appear to retain a conserved surface epitope because all Pseudomonas type IV pili recognize the same receptor on the host cell and cross-reactive antibodies specific for the receptor-binding site exist. Here, we present the crystallographic analysis of two crystal forms of truncated pilin from P. aeruginosa strain K122-4 (DeltaK122-4) at 1.54 and 1.8 A resolution, respectively. The DeltaK122-4 structure is compared to other crystallographically determined type IV pilin structures and an NMR structure of DeltaK122-4 pilin. A comparison with the structure of the highly divergent P. aeruginosa strain K (DeltaPAK) pilin indicates that the receptor-binding loop in both pilins forms a shallow depression with a surface that is formed by main-chain atoms. Conservation of this putative binding site is independent of the sequence as long as the main-chain conformation is conserved and could therefore explain the shared receptor specificity and antibody cross reactivity of highly divergent Pseudomonas type IV pilins.

Binding Sites↗

PubMed2003

Reciprocal transfer of class I MHC allele specificity between activating Ly-49P and Ly-49W receptors by exchange of beta 4-beta 5 loop residues.

Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2D(d), or H-2D(d) and D(k), respectively. In this report, we demonstrate that specificity for H-2D(k) can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the beta4-beta5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2D(k) recognition while still preserving H-2D(d) recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the beta4-beta5 loop, in determining class I MHC allele specificity.

Alleles↗

PubMed2003

When less is more: a more efficient vapour-diffusion protocol.

Reducing protein consumption during crystallization screening is of utmost importance to crystallographers because of the time, effort and money that goes into producing pure protein. One approach is to reduce sample volumes with robotics, but a patent and the high cost of equipment limits access. Here, it is shown that the same result can be obtained by reducing the sample concentration in a modified vapour-diffusion protocol, the dilution method. In this protocol, the protein and mother liquor in the crystallization drop are both diluted, while the mother liquor in the well remains undiluted. Vapour diffusion will shrink the initial volume of the crystallization drop, e.g. 1 micro l or more, to a drop size equivalent to one dispensed by a robot. This new crystallization method circumvents some of the current problems associated with robotic crystallization screening trials. Because of the large initial volume of the crystallization drop, the evaporation problem is eliminated and dispensing accuracy is improved. In addition, the likelihood that the crystallization experiment starts in the undersaturated region is increased.

Aldose-Ketose Isomerases↗

PubMed2003

Purification, crystallization and preliminary diffraction studies of the Pseudomonas aeruginosa strain K122-4 monomeric pilin.

The monomeric pilin from Pseudomonas aeruginosa strain K122-4 has been crystallized and preliminary X-ray diffraction data have been collected. Pilin is the monomeric subunit of the type IV pilus, the dominant adhesin of the opportunistic pathogen P. aeruginosa. The K122-4 pilin crystallizes as a dimer in space group P1, with unit-cell parameters a = 40.19, b = 38.93, c = 37.22 A, alpha = 66.38, beta = 111.12, gamma = 93.74 degrees. Diffraction data were collected using a synchrotron-radiation source and were processed to 1.54 A d-spacing.

Adhesins, Bacterial↗

PubMed2003

C-terminal amino acids of Helicobacter pylori alpha1,3/4 fucosyltransferases determine type I and type II transfer.

The alpha1,3/4 fucosyltransferase (FucT) enzyme from Helicobacter pylori catalyzes fucose transfer from donor GDP-beta-l-fucose to the GlcNAc group of two series of acceptor substrates in H. pylori lipopolysaccharide: betaGal1,3betaGlcNAc (Type I) or betaGal1,4betaGlcNAc (Type II). Fucose is added either in alpha1,3 linkage of Type II acceptor to produce Lewis X or in alpha1,4 linkage of Type I acceptor to produce Lewis A, respectively. H. pylori FucTs from different strains have distinct Type I or Type II substrate specificities. FucT in H. pylori strain NCTC11639 has an exclusive alpha1,3 activity because it recognizes only Type II substrates, whereas FucT in H. pylori strain UA948 can utilize both Type II and Type I acceptors; thus it has both alpha1,3 and alpha1,4 activity, respectively. To identify elements conferring substrate specificity, 12 chimeric FucTs were constructed by domain swapping between 11639FucT and UA948FucT and characterized for their ability to transfer fucose to Type I and Type II acceptors. Our results indicate that the C-terminal region of H. pylori FucTs controls Type I and Type II acceptor specificity. In particular, the highly divergent C-terminal portion, seven amino acids DNPFIFC at positions 347-353 in 11639FucT, and the corresponding 10 amino acids CNDAHYSALH at positions 345-354 in UA948FucT, controls the Type I and Type II acceptor recognition. This is the opposite of mammalian FucTs where acceptor preference is determined primarily by the N-terminal residues in the hypervariable stem domain.

Amino Acid Sequence↗

PubMed2002

Allelic variation in the ectodomain of the inhibitory Ly-49G2 receptor alters its specificity for allogeneic and xenogeneic ligands.

The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2D(d) and D(k) class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes D(d), and not D(k). The specificity for D(k) as well as D(d) of the wild-type Ly-49G(BALB/c) allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49G(BALB/c) allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49G(B6). Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.

Alleles↗