Biomedical subjects

H W van den Hooven

Publications and source records attributed to H W van den Hooven.

11 recordsLinked to original sources

PubMed2001

Disulfide bond structure of the AVR9 elicitor of the fungal tomato pathogen Cladosporium fulvum: evidence for a cystine knot.

Disease resistance in plants is commonly activated by the product of an avirulence (Avr) gene of a pathogen after interaction with the product of a matching resistance (R) gene in the host. In susceptible plants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor from the fungus Cladosporium fulvum induces defense responses in tomato plants carrying the Cf-9 resistance gene. This 28-residue beta-sheet AVR9 peptide contains three disulfide bridges, which were identified in this study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced, and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. After HPLC purification, the partially reduced peptides were sequenced to determine the positions of the modified cysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with free thiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteine modification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial) reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was further reduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially) reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstrating the importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing, it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridging pattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtained for AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor. However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knot fold is a commonly occurring motif with varying biological functions.

Alkylation↗

PubMed1999

Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.

The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.

Amino Acid Sequence↗

PubMed1998

Correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptide elicitors.

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.

Amino Acid Sequence↗

PubMed1997

Structural features of the final intermediate in the biosynthesis of the lantibiotic nisin. Influence of the leader peptide.

The antimicrobial membrane-interacting polypeptide nisin is a prominent member of the lantibiotic family, the members of which contain thioether-bridged residues called lanthionines. To gain insight into the complex biosynthesis and the structure/function relationship of lantibiotics, the final intermediate in the biosynthesis of nisin A was studied by nuclear magnetic resonance spectroscopy. In aqueous solution the leader peptide part of this precursor adopts predominantly a random coil structure, as does the synthetic leader peptide itself. The spatial structure of the fully modified nisin part of the precursor is similar to that of nisin in water. The leader peptide part does not interact with the nisin part of the precursor molecule. Thus, these two parts of the precursor do not influence each other's conformation significantly. The conformation of the precursor was also studied while complexed to micelles of dodecylphosphocholine, mimicking the primary target of the antimicrobial activity of nisin, i.e. the cytoplasmic membrane. The location of the molecule relative to the micelles was investigated by using micelle-inserted spin-labeled 5-doxylstearic acid. It was observed that the N-terminal half of the nisin part of the precursor interacts in a different way with micelles than does the corresponding part of mature nisin, whereas no significant differences were found for the C-terminal half of the nisin part. In this model system the leader peptide is in contact with the micelles. It is concluded that the strongly reduced in vivo activity of the precursor molecule relative to that of nisin is not caused by a difference in the spatial structure of nisin and of the corresponding part of precursor nisin in water or by a shielding of the membrane interaction surface of the nisin part of the precursor by the leader peptide. Probably a different interaction of the N-terminal part of the nisin region with membranes contributes to the low activity by preventing productive insertion. The residues of the leader peptide part just next to the nisin part are likely to contribute most to the low activity of the precursor.

Amino Acid Sequence↗

PubMed1997

The race-specific elicitor AVR9 of the tomato pathogen Cladosporium fulvum: a cystine knot protein. Sequence-specific 1H NMR assignments, secondary structure and global fold of the protein.

The secondary structure and global fold of the AVR9 elicitor protein of Cladosporium fulvum has been determined by 2D NMR and distance-geometry protocols. The protein consists of three anti-parallel strands forming a rigid region of beta-sheet. On the basis of the NMR-derived parameters and distance geometry calculations, it is evident that the AVR9 protein is structurally very homologuous to carboxy peptidase inhibitor (CPI) of which the X-ray structure is known. The AVR9 protein reveals the presence of a cystine knot, which consists of a ring formed by two disulfide bridges and the interconnecting backbone through which the third disulfide bridge penetrates. This structural motif is found in several small proteins such as proteinase inhibitors, ion channel blockers and growth factors. The implications of the structural relationship between AVR9 and other biologically active proteins are discussed.

Amino Acid Sequence↗

PubMed1996

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

Amino Acid Sequence↗

PubMed1995

Elucidation of the primary structure of the lantibiotic epilancin K7 from Staphylococcus epidermidis K7. Cloning and characterisation of the epilancin-K7-encoding gene and NMR analysis of mature epilancin K7.

Lantibiotics are bacteriocins that contain unusual amino acids such as lanthionines and alpha, beta-didehydro residues generated by posttranslational modification of a ribosomally synthesized precursor protein. The structural gene encoding the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was cloned and its nucleotide sequence was determined. The gene, which was named elkA, codes for a 55-residue preprotein, consisting of an N-terminal 24-residue leader peptide, and a C-terminal 31-residue propeptide which is posttranslationally modified and processed to yield mature epilancin K7. In common with the type-A lantibiotics nisin A and nisin Z, subtilin, epidermin, gallidermin and Pep5, pre-epilancin K7 has a so-called class-Al leader peptide. Downstream and upstream of the elkA gene, the starts of two open-reading-frames, named elkP and elkT, were identified. The elkP and elkT genes presumably encode a leader peptidase and a translocator protein, respectively, which may be involved in the processing and export of epilancin K7. The amino acid sequence of the unmodified pro-epilancin K7, deduced from the elkA gene sequence, is in full agreement with the amino acid sequence of mature epilancin K7, determined previously by means of NMR spectroscopy [van de Kamp, M., Horstink, L. M., van den Hooven, M. W., Konings, R. N. M., Hilbers, C. W., Sahl, H.-G., Metzger, J. W. & van de Ven, F. J. M. (1995) Eur. J. Biochem. 227, 757-771]. The first residue of mature epilancin K7 appears to be modified in a way that has not been described for any other lantibiotic so far. NMR experiments show that the elkA-encoded serine residue at position +1 of pro-epilancin K7 is modified to a 2-hydroxypropionyl residue in the mature protein.

Amino Acid Sequence↗

PubMed1995

Mechanistic studies of lantibiotic-induced permeabilization of phospholipid vesicles.

Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from PC liposomes in a delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold delta psi requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the lipid bilayer upon the imposition of a delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

Anti-Bacterial Agents↗

PubMed1995

Sequence analysis by NMR spectroscopy of the peptide lantibiotic epilancin K7 from Staphylococcus epidermidis K7.

The amino acid sequence of the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was determined by NMR spectroscopy. NMR spectroscopy was used because sequencing by conventional Edman degradation techniques was prohibited by internal sequence blocks owing to the presence of modified residues. Epilancin K7 consists of 31 residues, including two alpha,beta-didehydroalanine (one-letter code U) and two alpha,beta-didehydrobutyrine (O) residues, one lanthionine (A-S-A), two beta-methyllanthionines (A*-S-A), and six lysines. Epilancin K7 has a molecular mass of 3032 +/- 1.5 Da. The amino acid sequence of epilancin K7 was derived from both through-space dipolar proton-proton interactions and through-bond scalar proton-carbon interactions as detected by two-dimensional 1H-NOESY, 1H-ROESY and three-dimensional 1H-TOCSY-NOESY, and by two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy, respectively. The sequence is as follows: [sequence: see text] The N-terminal residue X partly resembles an alanine but its exact nature is unclear. The organization of the sulfide-bridge-containing (beta-methyl-)lanthionines was revealed by 1H-NMR and 1H,13C-NMR spectroscopy. Epilancin K7 has a linear structure and a high positive net charge, and therefore is classified as a type-A lantibiotic. NMR analysis of a degraded though still active form of epilancin K7 showed that two N-terminal residues of epilancin K7 were missing, owing to decomposition at the alpha,beta-didehydro alanine at position 3; it was called the epilancin K7-(3-31)-peptide (peptide fragment of epilancin K7 consisting of positions 3-31). The usefulness of three-dimensional 1H-TOCSY-NOESY, and two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy at natural abundance for the study of (modified) polypeptides is demonstrated.

Amino Acid Sequence↗

PubMed1993

NMR and circular dichroism studies of the lantibiotic nisin in non-aqueous environments.

The lantibiotic, nisin, which is known to interact with membranes of certain Gram-positive bacteria, was studied in three model systems which mimic a membrane-like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non-aqueous environments, at 40 degrees C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non-aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N-terminus.

Amino Acid Sequence↗

PubMed1990

Extreme differences in charge changes during protein evolution.

The maintenance of a proper distribution of charged amino acid residues might be expected to be an important factor in protein evolution. We therefore compared the inferred changes in charge during the evolution of 43 protein families with the changes expected on the basis of random base substitutions. It was found that certain proteins, like the eye lens crystallins and most histones, display an extreme avoidance of changes in charge. Other proteins, like phospholipase A2 and ferredoxin, apparently have sustained more charged replacements than expected, suggesting a positive selection for changes in charge. Depending on function and structure of a protein, charged residues apparently can be important targets for selective forces in protein evolution. It appears that actual biased codon usage tends to decrease the proportion of charged amino acid replacements. The influence of nonrandomness of mutations is more equivocal. Genes that use the mitochondrial instead of the universal code lower the probability that charge changes will occur in the encoded proteins.

Biological Evolution↗