Three-dimensional structure of diferric bovine lactoferrin at 2.8 A resolution

The three-dimensional structure of diferric bovine lactoferrin (bLf) has been determined by X-ray crystallography in order to investigate the factors that influence iron binding and release by transferrins. The structure was solved by molecular replacement, using the coordinates of diferric human lactoferrin (hLf) as a search model, and was refined with data to 2.8 A resolution by simulated annealing (X-PLOR) and restrained least squares (TNT). The final model comprises 5310 protein atoms (residues 5 to 689), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), 2 Fe3+, 2 CO32- and 50 water molecules. This model gives an R-factor of 0.232 for 21440 reflections in the resolution range 30.0 to 2.8 A. The folding of the bLf molecule is essentially the same as that of hLf, but bLf differs in the extent of closure of the two domains of each lobe, and in the relative orientations of the two lobes. Differences in domain closure are attributed to amino acid changes in the interface, and differences in lobe orientations to slightly altered packing of two hydrophobic patches between the lobes. Changed interdomain interactions may explain the lesser iron affinity of bLf, compared with hLf, and two lysine residues behind the N-lobe iron site of bLf offer new insights into the "dilysine trigger" mechanism proposed for iron release by transferrins. The bLf structure is also notable for several well-defined oligosaccharide units which demonstrate the structural factors that stabilise carbohydrate structure. One glycan chain, attached to Asn545, appears to contribute to interdomain interactions and may modulate iron release from the C-lobe.     

Refined structure of dimeric diphtheria toxin at 2.0 A resolution

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.     

Crystal structure of horseradish peroxidase C at 2.15 A resolution

The crystal structure of horseradish peroxidase isozyme C (HRPC) has been solved to 2.15 A resolution. An important feature unique to the class III peroxidases is a long insertion, 34 residues in HRPC, between helices F and G. This region, which defines part of the substrate access channel, is not present in the core conserved fold typical of peroxidases from classes I and II. Comparison of HRPC and peanut peroxidase (PNP), the only other class III (higher plant) peroxidase for which an X-ray structure has been completed, reveals that the structure in this region is highly variable even within class III. For peroxidases of the HRPC type, characterized by a larger FG insertion (seven residues relative to PNP) and a shorter F' helix, we have identified the key residue involved in direct interactions with aromatic donor molecules. HRPC is unique in having a ring of three peripheral Phe residues, 142, 68 and 179. These guard the entrance to the exposed haem edge. We predict that this aromatic region is important for the ability of HRPC to bind aromatic substrates.     

Canine parvovirus capsid structure, analyzed at 2.9 A resolution

The DNA-containing capsid of canine parvovirus (CPV) is analyzed following atomic refinement at 2.9 A resolution. The capsid contains 60 copies of the capsid protein related by icosahedral symmetry. The atomic model has been extended from the first residue (Gly37) of the unrefined 3.25 A structure towards the N terminus. The electron density shows that approximately 87% of the capsid proteins have N termini on the inside of the capsid, but for approximately 13%, the polypeptide starts on the outside and runs through one of the pores surrounding each 5-fold axis, explaining apparently conflicting antigenic data. Analysis of potential hydrogen bonds reveals approximately 50% more secondary structure than previously apparent. Most of the additional secondary structure are in the 71 and 221 residue-long loop insertions between beta-strands E and F and G and H, forming subunit-bridging sheets that likely add specificity to assembly interactions. Structural analysis of the extensive subunit interactions around the 3-fold axes shows that assembly is a multistep process with loops intertwining following initial contact. Estimated free energies of association suggest that the formation of 3 and 5-fold contacts likely takes precedence over 2-fold interactions. Energies for initial association into trimers or pentamers would be similar, but the intertwining of loops about the 3-fold axis adds an additional large activation barrier to dissociation. Analysis of the surfaces of the assembled capsid shows a surprising lack of basic amino acids that might have been expected to interact with the negatively charged phosphoribose backbone of the DNA. Instead, uncharged polar and van der Waal's interactions predominate in the packaging of single-stranded DNA into the capsid.     

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C-terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 A resolution. The structure reveals a compact fold distantly related to the C-type lectin carbohydrate recognition domain and the hyaluronan-binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.     

Crystal structure of the nucleosome core particle at 2.8 A resolution

The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.     

Crystal structure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution

The rate-limiting step in cholesterol biosynthesis in mammals is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a four-electron oxidoreductase that converts HMG-CoA to mevalonate. The crystal structure of HMG-CoA reductase from Pseudomonas mevalonii was determined at 3.0 angstrom resolution by multiple isomorphous replacement. The structure reveals a tightly bound dimer that brings together at the subunit interface the conserved residues implicated in substrate binding and catalysis. These dimers are packed about a threefold crystallographic axis, forming a hexamer with 23 point group symmetry. Difference Fourier studies reveal the binding sites for the substrates HMG-CoA and reduced or oxidized nicotinamide adenine dinucleotide [NAD(H)] and demonstrate that the active sites are at the dimer interfaces. The HMG-CoA is bound by a domain with an unusual fold, consisting of a central alpha helix surrounded by a triangular set of walls of beta sheets and alpha helices. The NAD(H) is bound by a domain characterized by an antiparallel beta structure that defines a class of dinucleotide-binding domains.     

Crystal structure of yellow meal worm alpha-amylase at 1.64 A resolution

The three-dimensional structure of the alpha-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques using diffraction data of a crystal of space group P212121 (a=51.24 A; b=93.46 A; c=96.95 A). The structure has been refined to a crystallographic R-factor of 17.7% for 58,219 independent reflections in the 7.0 to 1.64 A resolution range, with root-mean-square deviations of 0.008 A for bond lengths and 1.482 degrees for bond angles. The final model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density confirms that the N-terminal glutamine residue has undergone a post-transitional modification resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the first three-dimensional model of an insect alpha-amylase. The monomeric enzyme exhibits an elongated shape approximately 75 Ax46 Ax40 A and consists of three distinct domains, in line with models for alpha-amylases from microbial, plant and mammalian origin. However, the structure of TMA reflects in the substrate and inhibitor binding region a remarkable difference from mammalian alpha-amylases: the lack of a highly flexible, glycine-rich loop, which has been proposed to be involved in a "trap-release" mechanism of substrate hydrolysis by mammalian alpha-amylases. The structural differences between alpha-amylases of various origins might explain the specificity of inhibitors directed exclusively against insect alpha-amylases.     

Crystallization and structure determination of bovine profilin at 2.0 A resolution

Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(L-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69.15 A, b = 34.59 A, c = 52.49 A; alpha = gamma = 90 degrees, beta = 92.56 degrees) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2.55 A structure of profilin complexed to beta-actin. The refined model of bovine profilin has a crystallographic R-factor of 16.5% in the resolution range 6.0 to 2.0 A and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns. The structure of free bovine profilin is similar to that of bovine profilin complexed to beta-actin, and C alpha atoms from the two structures superimpose with an r.m.s. deviation of 1.25 A. This value is reduced to 0.51 A by omitting Ala1 and the N-terminal acetyl group, which lie at a profilin-actin interface in crystals of the complex. These residues display a strained conformation in crystalline profilin-actin but may allow the formation of a hydrogen bond between the N-acetyl carbonyl group of profilin and the phenol hydroxyl group of Tyr188 in actin. Several other actin-binding residues of profilin show different side-chain rotomer conformations in the two structures. The polypeptide fold of bovine profilin is generally similar to those observed by NMR for profilin from other sources, although the N terminus of Acanthamoeba profilin isoform I lies in a distorted helix and the C-terminal helix is less tilted with respect to the strands in the central beta-pleated sheet than is observed in bovine profilin. The majority of the aromatic residues in profilin are exposed to solvent and lie in either of two hydrophobic patches, neither of which takes part in an interface with actin. One of these patches is required for binding poly(L-proline) and contains an aromatic cluster comprising the highly conserved residues Trp3, Tyr6, Trp31 and Tyr139. In forming this cluster, Trp31 adopts a sterically strained rotamer conformation.(ABSTRACT TRUNCATED AT 400 WORDS)