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R Kaptein

Publications and source records attributed to R Kaptein.

At least 55 records · Page 3Linked to original sources
PubMed1997

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Amino Acid Sequence↗
PubMed1997

Involvement of laser photo-CIDNP (chemically induced dynamic nuclear polarization)-reactive amino acid side chains in ligand binding by galactoside-specific lectins in solution.

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single-site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.

Amino Acid Sequence↗
PubMed1997

The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif.

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 A for the backbone atoms and 1.12 A for all atoms for residues 6-36 and 46-67. The structure of IF1 is characterized by a five-stranded beta-barrel. The loop connecting strands three and four contains a short 3(10) helix but this region shows considerably higher flexibility than the beta-barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer-binding motif. Several proteins of this family are nucleic acid-binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.

Amino Acid Sequence↗
PubMed1997

Backbone and side chain dynamics of lac repressor headpiece (1-56) and its complex with DNA.

The dynamics of the backbone and (some of) the side chains of lac headpiece (1-56; lac HP56) have been studied for the free protein and for its complex with lac half-operator DNA by 15N T1 and T1p relaxation measurements combined with [1H-15N] NOE experiments. For the structurally well-defined part of the free lac HP56 (i.e., residues 3-49) a rigid backbone was found for residues in the three alpha-helices and for the turn of the helix-turn-helix motif. The loop between helices II and III of lac headpiece, which was characterized by slight disorder in the structure calculations, shows increased mobility. The detected side chains are very mobile. These data are in full agreement with the rms deviations in the structural data of free lac HP56. When lac HP56 is complexed with DNA, several changes in mobility take place. The most remarkable change was found for the loop region between helices II and III: residue His-29 within this loop interacts with Thy-3 of the operator DNA. As a result this mobile loop adapts itself to the DNA and becomes more rigid. Moreover, most DNA-contacting side chains show a significant decrease in flexibility, although these side chains do not become as rigid as the backbone. These results suggest that the mobility of the regions within lac HP56 important for complexation, i.e., the loop and the DNA-contacting side chains, is essential for a good fit onto the counterparts of the target DNA.

Bacterial Proteins↗
PubMed1997

Comparison between intact and desialylated human serum amyloid P component by laser photo CIDNP (chemically induced dynamic nuclear polarization) technique: an indication for a conformational impact of sialic acid.

The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is generated by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption- or emission-signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these amino acid residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one Trp/His-characteristic signal is decreased by 0.1 ppm. One Trp/His-signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The Tyr signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N-glycan of each monomer apparently affects surface presentation of distinct CIDNP-reactive amino acids of SAP [1]. A conformational change of the protein part of SAP in relation with a different orientation of the desialylated oligosaccharide chain in comparison to the complete one is a possible explanation of our CIDNP results.

Carbohydrate Conformation↗
PubMed1997

Carbohydrate-protein interaction studies by laser photo CIDNP NMR methods.

The side chains of tyrosine, tryptophan and histidine are able to produce CIDNP (Chemically Induced Dynamic Nuclear Polarization) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies surface accessibility of the respective groups to the light-absorbing dye. In principle, this technique allows the monitoring of the effect of ligand binding to a receptor and of site-directed mutagenesis on conformational aspects of any protein if CIDNP-reactive amino acids are involved. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial model study for several N-acetyl-glucosamine-binding lectins of increasing structural complexity as well as for a wild type bacterial sialidase and its mutants. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate is bound, CIDNP signals of side chain protons of tyrosine, tryptophan or histidine residues can be broadened and of reduced intensity. This is the case for hevein, pseudo-hevein, the four hevein domains-containing lectin wheat germ agglutinin (WGA) and the cloned B-domain of WGA 1 (domB) representing one hevein domain. This response indicates either a spatial protection by the ligand or a ligand-induced positioning of formerly surface-exposed side chains into the protein's interior part, thereby precluding interaction with the photo-activated dye. Some signals of protons from the reactive side chains can even disappear when the lectin-ligand complexes are monitored. The ligand binding, however, can apparently also induce a conformational change in a related lectin that causes the appearance of a new signal, as seen for Urtica dioica agglutinin (UDA) which consists of two hevein domains. Additionally, the three CIDNP-reactive amino acids are used as sensors for the detection of conformational changes caused by pH variations or by deliberate amino acid exchanges, as determined for the isolectins hevein and pseudo-hevein as well as for the cloned small sialidase of Clostridium perfringens and two of its mutants. Therefore, CIDNP has proven to be an excellent tool for protein-carbohydrate binding studies and can be established in glycosciences as a third biophysical method beside X-ray-crystallography and high-resolution multidimensional NMR studies which provides reliable information of certain structural aspects of carbohydrate-binding proteins in solution.

Carbohydrates↗
PubMed1997

Role of aromatic amino acids in carbohydrate binding of plant lectins: laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins.

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light-absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP-reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR- or X-ray crystallographic data permit a decision of the validity of the CIDNP method-derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling.

Amino Acids↗
PubMed1996

Solution structure of the immunodominant region of protein G of bovine respiratory syncytial virus.

The three-dimensional solution structure of the immunodominant central conserved region of the attachment protein G (BRSV-G) of bovine respiratory syncytial virus has been determined by nuclear magnetic resonance (NMR) spectroscopy. In the 32-residue peptide studied, 19 residues form a small rigid core composed of two short helices, connected by a type I' turn, and linked by two disulfide bridges. This unique fold is among the smallest stable tertiary structures known and could therefore serve as an ideal building block for the design of de novo proteins and as a test case for modeling studies. A characteristic hydrophobic pocket, lined by conserved residues, lies at the surface of the peptide and may play a role in receptor binding. This work provides a structural basis for further peptide vaccine development against the severe diseases associated with the respiratory syncytial viruses in both cattle and man.

Amino Acid Sequence↗
PubMed1996

NMR studies of the free alpha subunit of human chorionic gonadotropin. Structural influences of N-glycosylation and the beta subunit on the conformation of the alpha subunit.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Asn52 of its alpha subunit (alpha hCG) is essential for signal transduction, whereas the N-glycan at Asn78 stabilizes the structure of the protein. In this study, an almost complete 1H-NMR and a partial 13C-NMR spectral assignment for the amino acids and the N-glycans of alpha hCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of alpha hCG is solution, which was determined based on NOE data, is partially similar to that of the alpha subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33-58. In the crystal structure of hCG, residues 33-37 and 54-58 of the alpha subunit are part of an intersubunit seven-stranded beta-barrel and residues 41-47 constitute a 3(10)-helix. In contrast, in free alpha hCG in solution, amino acids 33-58 are part of a large disordered loop, indicating that in intact hCG interactions with the beta subunit of hCG stabilize the conformation of the alpha subunit. The NMR data of alpha hCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc-1 does not notably affect the conformation of the protein part. However, numerous 1H-NOEs between the GlcNAc-1 residue at Asn78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the alpha subunit. 1H-NOEs across the glycosidic linkages of the glycan, resonance-line widths, and 1H and 13C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn52 are largely extended in solution.

Amino Acid Sequence↗
PubMed1996

Refined structure of lac repressor headpiece (1-56) determined by relaxation matrix calculations from 2D and 3D NOE data: change of tertiary structure upon binding to the lac operator.

The solution structure of the DNA binding domain of lac repressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy. The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 phi and 15 chi 1 dihedral angle restraints. The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR. The final set of 32 selected structures displayed an r.m.s. deviation from the average of 0.43(+/-0.08) A angstroms (backbone) and 0.95(+/-0.08) angstroms (all heavy atoms) for the best defined region of the protein (residues 3 to 49). The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data. The structures revealed good stereochemical qualities. The conformations of the NMR structures of free and DNA complexed lac repressor headpiece were compared. The regions comprising the secondary structure elements show close correspondence for both conformations. However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece. This change in the conformation of the loop in lac HP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lac operator DNA. Finally, the lac headpiece residues that are intolerant to mutations were analysed. Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA.

Crystallography, X-Ray↗
PubMed1996

Structure of the LexA repressor-DNA complex probed by affinity cleavage and affinity photo-cross-linking.

The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage. These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator. In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein. In all four cases, the original amino acid was an arginine. The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as inferred from the structure of the LexA DNA binding domain [Fogh et al. (1994) EMBO J. 13, 3936-3944]. In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA. The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid. To reduce hydroxyl radical diffusion, the EDTA-iron cleavage reactions were done in the presence of high amounts of glycerol. The results indicate that amino acids 7 and 52 are near nucleotide pairs 8-12 of the operator and that amino acids 28 and 36 of LexA are near nucleotide pairs 5-8 of the operator. The results unambiguously define the orientation of the LexA DNA binding domain relative to the operator and provide support for the model of the LexA-operator complex proposed by Knegtel et al. [(1995) Proteins 21, 226-236]. Ethylation interference experiments further suggest that Arg-7 contacts the phosphate group between nucleotides 8 and 9 as predicted by the model.

Affinity Labels↗
PubMed1996

Affinity selective isolation of ligands from peptide libraries through display on a lac repressor "headpiece dimer".

DNA binding by the Escherichia coli lac repressor is mediated by the approximately 60 amino acid residue 'headpiece' domain. The dimer of headpiece domains that binds to the lac operator is normally formed by association of the much larger approximately 300 amino acid residue C-terminal domain. We have used in vitro selection to isolate 'headpiece dimer' molecules containing two headpiece domains connected via a short peptide linker. These proteins bind plasmid molecules with sufficient stability to allow association of a peptide epitope displayed at the C terminus of the headpiece dimer with the plasmid encoding that peptide. Libraries of peptides displayed on the C terminus of a headpiece dimer can be screened for specific receptor ligands by affinity enrichment of peptide-headpiece dimer-plasmid complexes using an immobilized receptor. After each round of enrichment, transformation of E. coli with recovered plasmids permits amplification of the selected population. After several rounds of enrichment, sequencing of individual clones reveals the structure of the selected peptides. Headpiece dimer libraries allow selection of peptide ligands of higher average affinity than similar libraries based on the intact lac repressor. Interestingly, the presence of the lac operator is not required for plasmid binding by the headpiece dimer protein.

Amino Acid Sequence↗
PubMed1996

Structure and dynamics of the DNA binding protein HU from Bacillus stearothermophilus by NMR spectroscopy.

The DNA-binding protein HU from Bacillus stearothermophilus (HUBst) is a dimer with a molecular weight of 195 kDa that is capable of bending DNA. An x-ray structure has been determined previously [Tanaka et al. 1984) Nature, vol. 310, pp. 376-381], but no structure could be established for a large part of the supposed DNA-binding beta-arms. Distance geometry and restrained molecular dynamics using nmr restraints were used to generate a set of 25 structures. These structures display a backbone rms deviation (RMSD) of 0.36 A for the well-defined region (residues 2-54 and 75-90). The structure of the core is very similar to that observed in the x-ray structure, with a pairwise RMSD of 1.06 A. The structure of the beta-hairpin arm contains a double flip-over at the prolines in the two strands of the beta-arm. Heteronuclear 15N relaxation measurements indicate that the beta-arm and the tip of the beta-arm is flexible. This explains the disorder observed in the solution and x-ray structures of the beta-arm with respect to the core of the protein. Overlayed onto itself the beta-arm is better defined, with a backbone RMSD of 1.0 A calculated for residues 54-59 and 69-74. The tip of the arm adopts a well-defined 4 : 6 beta-hairpin conformation. Changes in amide 15N and 1H chemical shifts upon titrating DNA are most pronounced for the residues in the beta-hairpin arm and for the residues in the second half of the third alpha-helix. Heteronuclear 15N relaxation data for free and complexed HUBst show that that the arms become structured upon DNA binding. Together with chemically induced nuclear polarization measurements on a mutant HUBst (M69Y; V76Y) this shows that the beta-hairpin arm is involved in direct DNA interaction.

Bacterial Proteins↗
PubMed1996

Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: application to HU protein from Bacillus stearothermophilus.

A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment. In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra. Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence. The range of NOE-graph templates of secondary structure motifs, incorporating alpha-helices and beta-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins. The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy. The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (13C, 15N) heteronuclear single quantum correlation spectrum of the (alpha + beta) type protein HU from Bacillus stearothermophilus. The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU. In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the beta-arm region (residues 53-76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the beta-arm at Ala57 and Pro72, respectively. At these residues, both strands of the beta-arm appear to flip inside out before continuing as a regular antiparallel beta-sheet.

Amino Acid Sequence↗
PubMed1996

AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR.

The AQUA and PROCHECK-NMR programs provide a means of validating the geometry and restraint violations of an ensemble of protein structures solved by solution NMR. The outputs include a detailed breakdown of the restraint violations, a number of plots in PostScript format and summary statistics. These various analyses indicate both the degree of agreement of the model structures with the experimental dat, and the quality of their geometrical properties. They are intended to be of use both to support ongoing NMR structure determination and in the validation of the final results.

Amino Acid Sequence↗
PubMed1995

Solution structure of the sequence-specific HMG box of the lymphocyte transcriptional activator Sox-4.

Two groups of HMG box proteins are distinguished. Proteins in the first group contain multiple HMG boxes, are non-sequence-specific, and recognize structural features as found in cruciform DNA and cross-over DNA. The abundant chromosomal protein HMG-1 belongs to this subgroup. Proteins in the second group carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. A solution structure for the non-sequence-specific C-terminal HMG box of HMG-1 has recently been proposed. Now, we report the solution structure of the sequence-specific HMG-box of the SRY-related protein Sox-4. NMR analysis demonstrated the presence of three alpha-helices (Val10-Gln22, Glu30-Leu41 and Phe50-Tyr65) connected by loop regions (Ser23-Ala49 and Leu42-Pro49). Helices I and II are positioned in an antiparallel mode and form one arm of the HMG box. Helix III is less rigid, makes an average angle of about 90 degrees with helices I and II, and constitutes the other arm of the molecule. As in HMG1B, the overall structure of the Sox-4 HMG box is L-shaped and is maintained by a cluster of conserved, mainly aromatic residues.

Amino Acid Sequence↗