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Zbigniew Dauter

Publications and source records attributed to Zbigniew Dauter.

At least 19 recordsLinked to original sources
PubMed2004

Radiation-damage-induced phasing with anomalous scattering: substructure solution and phasing.

Substructure-solution and phasing procedures using a combination of anomalous scattering and radiation-damage-induced isomorphous differences have been investigated. The tyrosine residues in thaumatin were iodinated with N-iodosuccinimide in the crystalline form as well as prior to crystallization. Several data sets were collected from both forms and used for substructure solution and phasing using various protocols, employing anomalous, isomorphous or both these signals. It was shown that combination of the anomalous and isomorphous signals in the form of the RIPAS (radiation-damage-induced phasing with anomalous scattering) strategy is beneficial for both locating the substructure and subsequent phasing.

Crystallography, X-Ray↗
PubMed2004

Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2.

Three different strains of Streptomyces aureofaciens produce the homologous ribonucleases Sa, Sa2 and Sa3. The crystal structures of ribonuclease Sa (RNase Sa) and its complexes with mononucleotides have previously been reported at high resolution. Here, the structures of two crystal forms (I and II) of ribonuclease Sa2 (RNase Sa2) are presented at 1.8 and 1.5 A resolution. The structures were determined by molecular replacement using the coordinates of RNase Sa as a search model and were refined to R factors of 17.5 and 15.0% and R(free) factors of 21.8 and 17.2%, respectively. The asymmetric unit of crystal form I contains three enzyme molecules, two of which have similar structures to those seen for ribonuclease Sa, with Tyr87 at the bottom of their active sites. In the third molecule, Tyr87 has moved substantially: the CA atom moves almost 5 A and the OH of the side chain moves 10 A, inserting itself into the active site of a neighbouring molecule at a similar position to that observed for the nucleotide base in RNase Sa complexes. The asymmetric unit of crystal form II contains two Sa2 molecules, both of which are similar to the usual Sa structures. In one molecule, two main-chain conformations were modelled in the alpha-helix. Finally, a brief comparison is made between the conformations of the Sa2 molecules and those of 34 independent molecules taken from 20 structures of ribonuclease Sa and two independent molecules taken from two structures of ribonuclease Sa3 in various crystal forms.

Amino Acid Sequence↗
PubMed2004

Structural effects of radiation damage and its potential for phasing.

A detailed analysis of radiation-damage-induced structural and intensity changes is presented on the model protein thaumatin. Changes in reflection intensities induced by irradiation display a parabolic character. The most pronounced structural changes observed were disulfide-bond breakage and associated main-chain and side-chain movements as well as decarboxylation of aspartate and glutamate residues. The structural changes induced on the sulfur atoms were successfully used to obtain high-quality phase estimates via an RIP procedure. Results obtained with ACORN suggest that the contribution originating from the partial structure may play an important role in phasing even at less than atomic resolution.

Aspartic Acid↗
PubMed2004

Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function.

BACKGROUND: The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. RESULTS: The structure was determined at 1.0 A resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. CONCLUSION: Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

Arsenite Transporting ATPases↗
PubMed2004

The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33 chaperone and its implications for the activation mechanism.

The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33.

Amino Acid Sequence↗
PubMed2004

The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle.

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.

Blotting, Western↗
PubMed2003

The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site.

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.

ATP-Dependent Proteases↗
PubMed2003

Twinned crystals and anomalous phasing.

Merohedral or pseudomerohedral twinning of crystals cannot be identified from inspection of the diffraction patterns. Several methods for the identification of twinning and the estimation of the twin fraction are suitable for macromolecular crystals and all are based on the statistical properties of the measured diffraction intensities. If the crystal twin fraction is estimated and is not too close to 0.5, the diffraction data can be detwinned; that is, related to the individual crystal specimen. However, the detwinning procedure invariably introduces additional inaccuracies to the estimated intensities, which substantially increase when the twin fraction approaches 0.5. In some cases, a crystal structure can be solved with the original twinned data by standard techniques such as molecular replacement, multiple isomorphous replacement or multiwavelength anomalous diffraction. Test calculations on data collected from a twinned crystal of gpD, the bacteriophage lambda capsid protein, show that the single-wavelength anomalous diffraction (SAD) method can be used to solve its structure even if the data set corresponds to a perfectly twinned crystal with a twin fraction of 0.5.

Animals↗
PubMed2003

Phasing on anomalous signal of sulfurs: what is the limit?

Recent years have witnessed significant advancements in X-ray data-acquisition techniques and phasing algorithms, which have made possible the successful use of a very small anomalous diffraction signal for the solution of crystal structures of macromolecules. Two crystal structures, a 44 kDa glucose isomerase containing nine sulfurs and a 33 kDa xylanase containing five sulfurs, have been solved from single-wavelength anomalous data using widely available methods and programs. These two enzymes contain less sulfur than most proteins in the bacterial or eukaryotic proteomes, providing a Bijvoet ratio of about 0.6%. For glucose isomerase the automatically interpretable electron-density maps could be obtained at high as well as low resolution. The S-SAD approach relies on the anomalous signal of sulfur naturally occurring in proteins and alleviates all need for sample derivatization. It may therefore be applicable to all protein crystals able to provide accurate diffraction data.

Aldose-Ketose Isomerases↗
PubMed2003

SAD manganese in two crystal forms of glucose isomerase.

Glucose isomerase from Streptomyces rubiginosus was crystallized in two forms: I222, with one molecule of 44 kDa in the asymmetric unit, and P2(1)2(1)2, with two unique molecules. The I222 structure is known, but the P2(1)2(1)2 form has not been solved before. X-ray diffraction data for the P2(1)2(1)2 form were collected at a wavelength of 1.54 A and data for the I222 form were collected at three different wavelengths: 1.34, 1.07 and 0.98 A. The amount of anomalous signal from one Mn and eight S atoms in these data sets varies from 1.24% to as low as 0.56%. The dual-space direct-methods program SHELXD, run against the Bijvoet differences, gave a clear solution of all anomalous scatterers for all data sets. The Mn positions only were used for SAD phasing of all four data sets. The electron-density map after density modification, resulting from the phasing of a single-wavelength data set and based purely on the anomalous deltaf" contribution, was clearly interpretable; an almost complete model of the protein was built by wARP without human intervention in all four cases. As far as is known, this is the first time that an anomalous signal as low as 0.6% has successfully been used to determine the structure of a macromolecule.

Aldose-Ketose Isomerases↗
PubMed2003

Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation.

Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp. strain GK16. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA. Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift. The autoproteolytic activation mechanism for GCA is described.

Amino Acid Sequence↗
PubMed2003

Purification and crystallization of the N-terminal domain from the human doublecortin-like kinase.

The unique doublecortin-like tandem of two homologous domains is found in certain microtubule-associated proteins such as doublecortin (DCX) and doublecortin-like kinase (DCLK). It is responsible for interactions with tubulin/microtubules and regulates microtubule dynamics. Here, the expression and purification of the tandem from human DCLK (residues 49-280) and of the isolated domains (residues 49-154 and 176-280) and the successful crystallization of the N-terminal domain (N-DCLK) are reported. High-quality wild-type crystals were obtained and a complete native data set was collected to 1.5 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 85.98, b = 29.62, c = 40.33 A, beta = 101.3 degrees. Crystals of SeMet-substituted N-DCLK (Leu120Met) were also obtained, but they exhibit the symmetry of space group P2(1), with unit-cell parameters a = 38.81, b = 29.43, c = 40.1 A, beta = 115.7 degrees.

Animals↗
PubMed2003

Phasing at high resolution using Ta6Br12 cluster.

The Ta(6)Br(12)(2+) cluster compound is known to be a powerful reagent for derivatization of crystals of large macromolecules at low resolution. The cluster is a regular octahedron of six Ta atoms with 12 bridging Br atoms at the edges of the octahedron. The cluster is compact, of approximately spherical shape, with a radius of about 6 A. Both tantalum and bromine display a significant anomalous diffraction signal at their absorption edges at 1.25 and 0.92 A, respectively. At resolutions lower than 5 A the tantalum cluster behaves as a super-atom and provides very large isomorphous and anomalous signals, which significantly diminish at about 4 A. However, beyond 3 A the individual Ta atoms can be resolved and the phasing power of the cluster increases again. The Ta(6)Br(12)(2+) cluster has been used for phasing four different proteins at high resolution. Ta(6)Br(12)(2+) appeared to be a mild derivatization reagent and, despite partial incorporation, led to a successful solution of crystal structures by the single-wavelength anomalous diffraction (SAD) approach.

Bromides↗
PubMed2003

Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein alpha.

CCAAT/enhancer-binding proteins (C/EBPs) are basic region leucine zipper (bZIP) transcription factors that regulate cell differentiation, growth, survival, and inflammation. To understand the molecular basis of DNA recognition by the C/EBP family we determined the x-ray structure of a C/EBPalpha bZIP polypeptide bound to its cognate DNA site (A(-5)T(-4)T(-3)G(-2)C(-1)G(1)C(2)A(3)A(4)T(5)) and characterized several basic region mutants. Binding specificity is provided by interactions of basic region residues Arg(289), Asn(292), Ala(295), Val(296), Ser(299), and Arg(300) with DNA bases. A striking feature of the C/EBPalpha protein-DNA interface that distinguishes it from known bZIP-DNA complexes is the central role of Arg(289), which is hydrogen-bonded to base A(3), phosphate, Asn(292) (invariant in bZIPs), and Asn(293). The conformation of Arg(289) is also restricted by Tyr(285). In accordance with the structural model, mutation of Arg(289) or a pair of its interacting partners (Tyr(285) and Asn(293)) abolished C/EBPalpha binding activity. Val(296) (Ala in most other bZIPs) contributes to C/EBPalpha specificity by discriminating against purines at position -3 and imposing steric restraints on the invariant Arg(300). Mutating Val(296) to Ala strongly enhanced C/EBPalpha binding to cAMP response element (CRE) sites while retaining affinity for C/EBP sites. Thus, Arg(289) is essential for formation of the complementary protein-DNA interface, whereas Val(296) functions primarily to restrict interactions with related sequences such as CRE sites rather than specifying binding to C/EBP sites. Our studies also help to explain the phenotypes of mice carrying targeted mutations in the C/EBPalpha bZIP region.

Amino Acid Sequence↗
PubMed2003

PDZ tandem of human syntenin: crystal structure and functional properties.

Syntenin, a 33 kDa protein, interacts with several cell membrane receptors and with merlin, the product of the causal gene for neurofibromatosis type II. We report a crystal structure of the functional fragment of human syntenin containing two canonical PDZ domains, as well as binding studies for full-length syntenin, the PDZ tandem, and isolated PDZ domains. We show that the functional properties of syntenin are a result of independent interactions with target peptides, and that each domain is able to bind peptides belonging to two different classes: PDZ1 binds peptides from classes I and III, while PDZ2 interacts with classes I and II. The independent binding of merlin by PDZ1 and syndecan-4 by PDZ2 provides direct evidence for the coupling of syndecan-mediated signaling to actin regulation by merlin.

Actins↗