Crystal structure of human prion protein bound to a therapeutic antibody.

Prion infection is characterized by the conversion of host cellular prion protein (PrP(C)) into disease-related conformers (PrP(Sc)) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP(C) rather than PrP(Sc). We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP(C) and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP(C) conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular beta-sheet. The importance of this residue in mediating protein-protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.

A critical assessment of the evidence from XAFS and crystallography for the breakage of the imidazolate bridge during catalysis in CuZn superoxide dismutase.

BACKGROUND: Copper-zinc superoxide dismutase (CuZn SOD) protects cells from the toxic effects of superoxide radicals. Key steps in the catalytic mechanism of CuZn SOD are thought to be the breakage of the imidazolate bridge between copper and zinc upon reduction of the copper site and the subsequent proton donation from the bridging histidine. This view has been recently challenged by a crystallographic study at 1.9 A resolution where evidence for a five-coordinate copper site in the reduced enzyme was provided. In contrast, a crystallographic study of yeast CuZn SOD at 1.7 A has confirmed the breaking of the bridging histidine in reduced crystals. We have examined the nature of the changes in metal coordination which result upon reduction of the enzyme using the X-ray absorption fine structure (XAFS) technique. RESULTS: The copper and zinc K-edge XAFS data of bovine SOD, recorded in the buffer systems used in the two crystallographic studies, were analyzed by constrained refinement using fast curved wave theory, taking full account of multiple-scattering effects. The study confirms that in the oxidized form of the enzyme the copper atom is five coordinate, with four histidine ligands at 1.99 +/- 0.02 A and a water molecule at 2.18 +/- 0.03 A. In the reduced form of the enzyme, one of the histidine ligands and the water molecule are lost from the inner coordination sphere of the copper, with the three remaining histidines ligated at 1.97 +/- 0.02 A. The X-ray absorption near edge structure (XANES) of the reduced enzyme is consistent with an approximate trigonal planar geometry at the copper site. The XAFS at the zinc K-edge is essentially identical in both the oxidized and reduced enzyme and is accounted for by three histidines coordinated at 2.01 +/- 0.02 A and an aspartate ligand at 1.96 +/- 0.03 A. CONCLUSIONS: The existence of a three-coordinate cuprous ion in bovine CuZn SOD is demonstrated and is a key feature of catalytic degradation of superoxide substrate by SOD involving alternate Cu(I) and Cu(II) states of the enzyme. Only subtle changes in the zinc K-edge XAFS take place upon reduction. Thus the reaction mechanism which involves breakage of the bridging histidine is unambiguously supported by the XAFS data.

Structure of bovine milk calcium phosphate determined by X-ray absorption spectroscopy.

Calcium in cow's milk is mainly in the form of calcium phosphate-phosphoprotein complexes known as casein micelles. These micelles, in contrast to other phosphoprotein complexes in bone and other tissues, can be readily isolated and studied, but conventional techniques have given ambiguous and conflicting evidence on the structure of milk calcium phosphate. Extended X-ray absorption fine structure and near-edge structure measurements at the newly commissioned Synchrotron Radiation Source at Daresbury indicate that it closely resembles brushite, CaHPO4 X 2H2O. This result, and chemical analysis, requires that phosphate groups from the matrix phosphoproteins be incorporated in the brushite lattice, probably in the surface, suggesting that these organic phosphate groups act as heterogeneous nucleation sites for phase separation of the calcium phosphate from solution.

The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS.

The EXAFS of the K-edge of copper in azurin from Pseudomonas aeruginosa has been measured in solutions of the oxidized and reduced protein, at both low and high pH. Model compounds of known molecular structure, exhibiting Cu-N and Cu-S bonds of varying length, were studied as well. The major shell of the high-pH oxidized azurin EXAFS contains contributions of two N(His) at 1.95 +/- 0.03 A, and one S(Cys) at 2.23 +/- 0.03 A. Some minor contributions from the carbon atoms of the histidine residues and the distal sulfur atom are observed in the 3-4 A region. Upon reduction a decrease is seen in amplitude of the main peak in the Fourier transform, due to a lengthening of one of the Cu-N(His) bonds (2.05 +/- 0.03 A), and a shortening of the other (1.89 +/- 0.03 A), both by approx. 0.1 A. Indications for a Cu-S(Met) bond are found in the reduced azurin data (2.70 +/- 0.05 A). However, in the oxidized protein, this bond could not be determined unambiguously, in line with results of a model compound featuring weak Cu-thioether coordination. The effect of pH is only slight for both the oxidized and the reduced protein, and no significant changes in bond lengths are found upon a change of pH from 4.1 to 9.1. The relevance of these findings for the interpretation of the existing data on the redox activity of the protein is discussed.

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal.

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.

Conformational variability of the Cu site in one subunit of bovine CuZn superoxide dismutase: the importance of mobility in the Glu119-Leu142 loop region for catalytic function.

The structure of the catalytic site in one subunit of bovine CuZn superoxide dismutase is shown to be highly variable. A series of crystal structures at approximately 1.7 A have been determined using data collected from different crystals. Several conformations are observed for the copper site from one of the subunits. These conformations lie between those expected for the Cu(II) and Cu(I) forms of the enzyme and may represent a slow positional rearrangement of the Cu site during the crystallisation process due to the presence of a trace reductant in the mother liquor. These states perhaps indicate some functionally relevant structural steps that ultimately result in the breakage of the imidazolate bridge between the two metal sites. This behaviour is not observed for the second subunit of the dimeric enzyme, which remains in the five-coordinate, distorted square planar geometry in all cases. We suggest that this asymmetric behaviour may be caused by the lack of mobility for the Glu119-Leu142 loop region in the second subunit caused by crystal contacts. This region forms one wall of the active-site cavity, and its mobility has been suggested, via molecular dynamics studies, to be important for the catalytic mechanism.

Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin.

Recent X-ray crystallographic and solution X-ray scattering studies have shown that transferrins (serum transferrin, lactoferrin and ovotransferrin) undergo a major conformational change when iron is incorporated into the molecule. Apo-proteins show a structure with open interdomain clefts which close when iron is bound. The closed conformation has been suggested as an important step in the receptor recognition. Here, we report X-ray solution scattering experiments of the mutated N-terminal fragment of human serum transferrin with Asp63-->Ser (Cys). The data provide the first direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft and that this trigger mechanism is disrupted by mutation of Asp63, the only ligand of iron from domain I.

Metal-induced conformational changes in transferrins.

Recent studies on iron-loaded transferrins have revealed a conformational change upon binding iron due to a domain closure. It has been suggested that the domain closure may be the key for the receptor recognition of the metal loaded transferrin (Grossmann et al., 1992). Small angle X-ray scattering has been used to provide direct structural information on the conformational changes that may take place upon the binding and release of different metals to the transferrins in solution. The data show that In3+ and Cu2+ induce the same domain closure as Fe3+; Al3+ causes a conformational change of somewhat smaller magnitude while Hf4+ does not induce any conformational change. The results are discussed in terms of the molecular recognition of metal loaded transferrin by the receptor.

The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase.

An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein. We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution. The biological significance of this first molecular structure of a nitrogenase complex is discussed.

X-ray structure of a blue-copper nitrite reductase in two crystal forms. The nature of the copper sites, mode of substrate binding and recognition by redox partner.

Denitrification is one of the main steps of the global nitrogen cycle that is sustained by prokaryotic organisms. Denitrifying bacteria use two entirely different enzymes in this process, one based on haem cd1 prosthetic groups and the other on type 1-type 2 Cu centres. Copper-containing nitrite reductases (NiRs) are sub-divided into blue and green NiRs, which are respectively thought to be redox partners of azurins and pseudo-azurins. Crystallographic structures of the blue nitrite reductase from Alcaligenes xylosoxidans (AxNiR) are presented in the oxidised hexagonal form and the substrate-bound orthorhombic form to 2.1 A and 2.8 A resolution, respectively. The complete amino acid sequence of AxNiR has been determined by conventional chemical analysis. A 3 A structure of AxNiR has been published where the modelling was based on the sequence of another blue NiR. The higher resolution of the hexagonal form together with the correct sequence allows a detailed comparison with the crystallographic structures of the green NiRs. There is a striking difference in the overall surface charge distribution between the two sub-groups, providing a neat structural explanation for their different reactivities to pseudoazurin or azurin and supporting the view that electron transfer proceeds via complex formation. A detailed examination of the type-1 Cu site, the site responsible for the colour, reveals several subtle differences, including a lateral displacement of 0.7 A for Smet. The structure of the type-2 Cu site, and changes that occur upon substrate binding are discussed in terms of the catalytic mechanism. The similarity of the type 2 Cu site to the catalytic Zn site in carbonic anhydrase and the catalytic Cu site of superoxide dismutase is re-examined in view of the high-resolution (2.1 A) structure.

Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase.

The crystallographic structures of several copper-containing nitrite reductases are now available. Despite this wealth of structural data, no definitive information is available as to whether the reaction proceeds by an ordered mechanism where nitrite binds to the oxidised type 2 site, followed by an internal electron transfer from the type 1 Cu, or whether binding occurs to the reduced type 2 Cu centre, or a random mechanism operates. We present here the first structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absorption spectroscopy. The reduced type 2 Cu site EXAFS shows striking similarity to the EXAFS data for reduced bovine superoxide dismutase (Cu2Zn2 SOD), providing strong evidence for the loss of the water molecule from the catalytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site similar to that in Cu2Zn2 SOD. The reduced type 2 Cu site of AxNiR is shown to be unable to bind inhibitory ligands such as azide, and to react very sluggishly with nitrite leading to only a slow re-oxidation of the the type 1 centre. These observations provide strong evidence that turnover of AxNiR proceeds by an ordered mechanism in which nitrite binds to the oxidised type 2 Cu centres before electron transfer from the reduced type 1 centre occurs. We propose that the two links between the Cu sites of AxNiR, namely His129-Cys130 and His89-Asp92-His94 are utilised for electron transfer and for communicating the status of the type 2 Cu site, respectively. Nitrite binding at type 2 Cu is sensed by the proton abstracting group Asp92 and the type 2 Cu ligand His94, and relayed to the type 1 Cu site via His89 thus triggering an internal electron transfer. The similarity of the type 2 Cu NiR catalytic site to the reduced Cu site of SOD is examined in some detail together with the biochemical evidence for the SOD activity of AxNiR.

The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants.

Ligand-induced conformational change in transferrins has been studied by site-directed mutagenesis of human serum half molecule (N-lobe), X-ray absorption fine structure (XAFS) spectroscopy and X-ray solution scattering. Use of recent advances in data analysis has been made for extracting model-independent molecular shapes from X-ray solution scattering data for the intact, the half molecule and its mutants. Clear evidence is provided that the transferrin molecule (intact as well as N-lobe), in its apo and holo forms, exists for the majority of the time in well-defined specific conformations representing the "fully opened" and "closed" states of the molecule, respectively. Evidence is also provided for the existence of an additional conformation, referred to here as the "intermediate" conformation for simplicity, which is trapped in the case of some of the mutants in the iron-bound form. We suggest that domain closure in the transferrin molecule is a two-step process, with the intermediate conformation representing the first stage of domain closure (approximately 20 degrees hinge-twist of domain II). Our data are not inconsistent with the ligand-free molecule sampling the closed states occasionally (< or = 10%) but are not in support of a continuous conformational search between the fully opened and closed states in the absence of iron.

XAFS study of the high-affinity copper-binding site of human PrP(91-231) and its low-resolution structure in solution.

Here, we describe the structure of a C-terminal high-affinity copper-binding site within a truncated recombinant human PrP containing residues 91-231, which lacks the octapeptide repeat region. We show that at least two extra co-ordinating groups are involved in binding this copper(II) ion in conjunction with histidine residues 96 and 111 in a region of the molecule known to be critical in conferring strain type. In addition, using X-ray solution scattering, a low-resolution shape of PrP(91-231) is provided. The restored molecular envelope is consistent with the picture where the N-terminal segment, residues 91-120, extends out from the previously known globular domain containing residues 121-231.

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.

The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene.

The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced. A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers. The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence. The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli.

Roxatidine versus ranitidine in the treatment of patients with duodenal ulcer: a randomized, double-masked, multicenter study.

The comparative efficacy of roxatidine and ranitidine in the treatment of patients with acute duodenal ulcer was assessed at 4 and 6 weeks in this multicenter study. Ninety-four of 192 patients were given roxatidine in a single nightly dose of 150 mg, and 98 patients were given ranitidine in a single nightly dose of 300 mg. All the patients had endoscopically proven duodenal ulcer. Of the 171 assessable patients, ulcers were healed in 88% of the roxatidine group (73 of 83) and in 84% of the ranitidine group (74 of 88). No serious adverse events were reported in either group. We conclude that roxatidine 150 mg once daily is as effective and safe for the treatment of acute duodenal ulcer as ranitidine 300 mg once daily.

EXAFS studies of the calcium salts of natural DNA and polydA:polydT.

Preliminary EXAFS experiments were carried out on film of the Ca salts of the synthetic polynucleotide polydA:polydT at 95%, 81%, and 76% relative humidity (r.h.) and for the Ca salt of chicken erythrocyte DNA at 81% r.h. (approximately 43% GC pairs). Detailed analysis of EXAFS data shows that the Ca2+ ion is in fairly close proximity (within 4 A) to a number of phosphorous atoms. This is in contradiction with the recently proposed model, which assumes a close coordination between the cations and the purine and pyrimidine bases deep inside the polynucleotide molecule, so that the distance to the nearest phosphorous atoms must not be less than 5 A. Instead, the EXAFS results suggest that the Ca2+ ions are, for the most part, located at the periphery of individual polydA:polydT (or DNA) molecules, possibly serving as intermolecular links.

EXAFS studies of the molybdenum center of xanthine oxidase.

EXAFS spectra associated with the K-absorption edge of molybdenum in the desulpho and functional forms of xanthine oxidase and some potential synthetic analogues have been obtained. These data indicate that the immediate environment of the molybdenum is different in the two forms of the enzyme and that desulpho xanthine oxidase resembles that in [MoO2(S2CNEt2)2] and [MoO2(ethylcysteine)2]. The cyanolysable sulphur atom of functional xanthine oxidase is suggested to be tightly bound to the molybdenum at a distance of less than or equal to 2.3 A.

Stereochemistry of iron in deoxyhaemoglobin.

The EXAFS of human deoxyhaemoglobin closely resembles that of a synthetic model in which the displacement of the iron from the mean porphyrin plane is 0.426 +/- 0.004 A, similar to the displacement of 0.56 +/- 0.03 A found by single crystal X-ray analysis of deoxyhaemoglobin. We find the same Fe-N of 2.06 +/- 0.01 A distance as Eisenberger et al., but show that the displacement of the iron from the nitrogen plane cannot be calculated from that distance.

X-ray crystallography and computational docking for the detection and development of protein-ligand interactions.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterised by the selective dysfunction and death of the upper and lower motor neurons. Median survival rates are between 3 and 5 years after diagnosis. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) have been linked to a subset of familial forms of ALS (fALS). Herein, we describe a fragment- based drug discovery (FBDD) approach for the investigation of small molecule binding sites in SOD1. X-ray crystallography has been used as the primary screening method and has been shown to directly detect protein-ligand interactions which cannot be unambiguously identified using other biophysical methods. The structural requirements for effective binding at Trp32 are detailed for a series of quinazoline-containing compounds. The investigation of an additional site that binds a range of catecholamines and the use of computational modelling to assist fragment evolution is discussed. This study also highlights the importance of ligand solubility for successful Xray crystallographic campaigns in lead compound design.