Rudolf Ladenstein

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray crystal structure of the blue oxidase ascorbate oxidase from Zucchini: Analysis of the polypeptide fold and a model of the copper sites and ligands

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.

THE THREE-DIMENSIONAL STRUCTURE OF CLASS PI GLUTATHIONE S-TRANSFERASE IN COMPLEX WITH GLUTATHIONE SULFONATE AT 2.3 A RESOLUTION

The three‐dimensional structure of class pi glutathione S‐transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1–74) consists of a central four‐stranded beta‐sheet flanked on one side by two alpha‐helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81–207) contains five alpha‐helices. The dimeric molecule is globular with dimensions of about 55 A ×52 A ×45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S‐transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.

Ion pairs and the thermotolerance of proteins from hyperthermophiles: a "traffic rule" for hot roads.

The proteins from hyperthermophilic organisms maintain their biologically active structure at temperatures that are significantly higher than the denaturation temperatures of their mesophilic counterparts. The fact that there is usually a high degree of sequence and structural homology between these two classes of proteins suggests that the source of this extreme thermal tolerance is hidden in the delicate balance of the non-covalent interactions. Among the large number of factors identified in the literature as being responsible for the thermostability of these proteins, this article focuses on electrostatic interactions. It demonstrates that the optimization of electrostatic interactions by increasing of the number of salt bridges is a driving force for enhancement of the thermotolerance of proteins from hyperthermophilic microorganisms. This feature is less evident in proteins from thermophilic organisms and is absent from mesophile-derived proteins.

Three-dimensional structure of class π glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 Å resolution☆

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitors glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

Structure analysis and molecular model of the selenoenzyme glutathione peroxidase at 2.8 A resolution.

Abstract The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole ( M r = 84,000), has been determined at 2.8 A resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar. The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of r ≈ 19 A . A tentative sequence corresponding to a partially refined model ( R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S 2 revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold. The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).

Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus.

The archaeon Sulfolobus solfataricus expresses large amounts of a small basic protein, Sso7d, which was previously identified as a DNA-binding protein possibly involved in compaction of DNA. We have determined the solution structure of Sso7d. The protein consists of a triple-stranded anti-parallel β-sheet onto which an orthogonal double-stranded β-sheet is packed. This topology is very similar to that found in eukaryotic Src homology-3 (SH3) domains. Sso7d binds strongly (Kd < 10 μM) to double-stranded DNA and protects it from thermal denaturation. In addition, we note that ɛ-mono-methylation of lysine side chains of Sso7d is governed by cell growth temperatures, suggesting that methylation is related to the heat-shock response.

Heavy riboflavin synthase from Bacillus subtilis: Crystal structure analysis of the icosahedral β60 capsid at 3·3 Å resolution☆

Geometric features as well as possible functional properties of the substrate binding sites at the pentamer interfaces are described. Ligand binding at the pentamer interface regions increases the stability of the beta 60 capsid considerably and influences the reassembly of isolated beta-subunits.

Extrinsic protein stabilization by the naturally occurring osmolytes β-hydroxyectoine and betaine

Abstract Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, β-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein. The osmolyte β-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer. At a concentration of 3 M it increased the melting temperature of RNase A (Tm) by more than 12 K and gave rise to a stability increase of 10.6 kJ/mol at room temperature. The heat capacity difference between the folded and unfolded state (ΔCp) was found to be significantly increased. Betaine stabilized RNase A only at concentrations less than 3 M. Also, here ΔCp was found to be increased. Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes. The significant stabilization of RNase A by β-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature.

The Multi-layered Structure of Dps with a Novel Di-nuclear Ferroxidase Center

The crystallization of cellular components represents a unique survival strategy for bacterial cells under stressed conditions. A highly ordered, layered structure is often formed in such a process, which may involve one or more than one type of bio-macromolecules. The main advantage of biocrystallization has been attributed to the fact that it is a physical process and thus is independent of energy consumption. Dps is a protein that crystallizes to form a multi-layered structure in starved cells in order to protect DNA against oxidative damage and other detrimental factors. The multi-layered crystal structure of a Dps protein from Bacillus brevis has been revealed for the first time at atomic resolution in the absence of DNA. Inspection of the structure provides the first direct evidence for the existence of a di-nuclear ferroxidase center, which possesses unique features among all the di-iron proteins identified so far. It constitutes the structural basis for the ferroxidase activity of Dps in the crystalline state as well as in solution. This finding proves that the enzymatic process of detoxification of metal ions, which may cause severe oxidative damage to DNA, is the other important aspect of the defense mechanism performed by Dps. In the multi-layered structure, Dps dodecamers are organized in a highly ordered manner. They adopt the classic form of hexagonal packing in each layer of the structure. Such arrangement results in reinforced structural features that would facilitate the attraction and absorption of metal ions from the environment. The highly ordered layered structure may provide an ideal basis for the accommodation of DNA between the layers so that it can be isolated and protected from harmful factors under stress conditions.