Rearrangement of Cruickshank's formulae for the diffraction-component precision index.

The formulae for the diffraction-component precision index introduced by Cruickshank [(1999), Acta Cryst. D55, 583-601] are simplified using two approximations. A rearranged formula for the precision index is presented which can readily be calculated from experimental data. It is shown that the precision varies as (resolution)(5/2) if R and completeness are maintained. It varies as (completeness of intensity measurement)(-5/6) and its dependence on the inclusion of solvent atoms is discussed.

Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator.

The Kunitz-type soybean trypsin inhibitor (STI) has played a key role in the early study of proteinases, having been used as the main substrate in the biochemical and kinetic work that led to the definition of the standard mechanism of action of proteinase inhibitors. A partial structure of STI complexed with porcine trypsin has previously been reported, in which the first 93 residues of the inhibitor, including the region of contact with trypsin, were relatively well defined, whereas for the remaining part of the peptide chain only some Calpha atoms were located. The structure of the inhibitor in its free form has now been determined by molecular replacement to 2.5 A, using the coordinates of the homologous Erythrina trypsin inhibitor as a search model. When the refined atomic coordinates of STI are compared with the partial model previously available, the conformation of the reactive-site loop and its position with respect to the main body of the molecule does not change when the inhibitor interacts with trypsin. There are instead, despite the high similarity in the overall tertiary structure, significant differences between STI and Erythrina trypsin inhibitor (ETI) in the region which is in contact with the enzyme in the STI:trypsin crystal structure. Some of these differences can explain the unique specificity of ETI and its ability to inhibit the fibrinolytic enzyme tissue-type plasminogen activator.

Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy.

BACKGROUND: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G2 (CPG2), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. RESULTS: We have determined the crystal structure of CPG2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. CONCLUSIONS: We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

Control of nucleation of protein crystals.

Control of nucleation may be needed to obtain a reliable supply of large protein crystals, when standard techniques give many small or twinned crystals. Heterogeneous nucleation may be controlled by the use of fine filters, with the elimination of airborne contaminants by working under paraffin oil. The area of contact with the supporting vessel also has an important effect. A heterogenous nucleant for lysozyme (identified earlier) has been shown to be effective for carboxypeptidase G2. Control of homogeneous nucleation (previously demonstrated by dilutions of a nucleating sample after various times of incubation) may also be achieved by incubating a sample at 1 temperature, where nucleation can occur, and changing the temperature to conditions where there is growth but no nucleation.

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2.

The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 x 0.3 x 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.

Crystal structure of cholesterol oxidase complexed with a steroid substrate: implications for flavin adenine dinucleotide dependent alcohol oxidases.

Cholesterol oxidase from Brevibacterium sterolicum is a flavin-dependent enzyme that catalyzes the oxidation and isomerization of 3 beta-hydroxy steroids with a double bond at delta 5-delta 6 of the steroid ring backbone. The crystal structure of the free enzyme in the absence of a steroid substrate has previously been determined. In this paper we report the crystal structure of the complex of cholesterol oxidase with the steroid substrate dehydroisoandrosterone, refined at 1.8-A resolution. The final crystallographic R-value is 15.7% for all reflections between 10.0- and 1.8-A resolution. The steroid is buried within the protein in an internal cavity which, in the free enzyme crystal structure, was occupied by a lattice of water molecules. The conformations of a number of side chains lining the active-site cavity have changed in order to accommodate the steroid substrate. A loop region of the structure between residues 70 and 90 differs significantly between the substrate-free and substrate-bound forms of the enzyme, presumably to facilitate binding of the steroid. The hydroxyl group of the steroid substrate is hydrogen-bonded to both the flavin ring system of the FAD cofactor and a bound water molecule. FAD-dependent cholesterol oxidase shares significant structural homology with another flavoenzyme, glucose oxidase, suggesting that it might also be a member of the glucose-methanol-choline (GMC) oxidoreductase family. Although there is only limited sequence homology, a superposition of these two structures reveals a conserved histidine residue within hydrogen-bonding distance of the active-site water molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces.

Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253-->Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.

Control of nucleation in the crystallization of lysozyme.

This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.

Molecular mechanics simulations of a conformational rearrangement of D-xylose in the active site of D-xylose isomerase.

A proposed reaction mechanism for the enzyme D-xylose isomerase involves the ring opening of the cyclic substrate with a subsequent conformational rearrangement to an extended open-chain form. Restrained energy minimization was used to simulate the rearrangement. In the ring-opening step, the substrate energy function was gradually altered from a cyclic to an open-chain form, with energy minimization after each change. The protein/sugar contact energy did not increase significantly during the process, showing that there was no steric hindrance to ring opening. The conformational rearrangement involves an alteration in the coordination of the substrate to metal ion [1], which was induced by gradually changing restraints on metal/ligand distances. By allowing varying amounts of flexibility in the protein and examining a simplified model system, the interactions of the sugar with metal ion [1] and its immediate ligands were found to be the most important contributors to the energy barrier for the change. Only small changes in the positions of protein atoms were required. The energy barrier to the rearrangement was estimated to be less than the Arrhenius activation energy for the enzymatic reaction. This is in accordance with experimental indications that the isomerization step is rate determining.

Structure and mechanism of D-xylose isomerase.

The action of xylose isomerase depends on the presence of two divalent cations. Crystal structure analyses of the free enzyme, and of the enzyme bound to a variety of substrates and inhibitors, have provided models for a number of distinct intermediates along the reaction pathway. These models, in turn, have suggested detailed mechanisms for the various chemical steps of the reaction: a ring opening catalysed by an activated histidine, a hydride-shift isomerization, and a ring closure which may be facilitated by a polarised water molecule.

Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution.

Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.

Many crystal forms of human immunodeficiency virus reverse transcriptase.

Many crystal forms of human immunodeficiency virus reverse transcriptase have been obtained by vapour diffusion, microbatch and microdialysis methods. Despite their apparent morphological perfection, no X-ray diffraction has been discernible in most cases with these crystals.

Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds.

The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.

Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift.

The active site and mechanism of D-xylose isomerase have been probed by determination of the crystal structures of the enzyme bound to various substrates, inhibitors and cations. Ring-opening is an obligatory first step of the reaction and is believed to be the rate-determining step for the aldose to ketose conversion. The structure of a complex with a cyclic thio-glucose has been determined and it is concluded that this is an analogue of the Michaelis complex. At -10 degrees C substrates in crystals are observed in the extended chain form. The absence of an appropriately situated base for either the cyclic or extended chain forms from the substrate binding site indicates that the isomerisation does not take place by an enediol or enediolate mechanism. Binding of a trivalent cation places an additional charge at the active site, producing a substrate complex that is analogous to a possible transition state. Of the two binding sites for divalent cations, [1] is permanently occupied under catalytic conditions and is co-ordinated to four carboxylate groups. In the absence of substrate it is exposed to solvent, and in the Michaelis complex analogue, site [1] is octahedrally coordinated, with ligands to O-3 and O-4 of the thiopyranose. In the complex with an open-chain substrate it remains octahedrally co-ordinated, with ligands to O-2 and O-4. Binding at a second cation site [2] is also necessary for catalysis and this site is believed to bind Co2+ more strongly than site [1]. This site is octahedrally co-ordinated to three carboxylate groups (bidentate co-ordination to one of them), an imidazole and a solvent molecule. It is proposed that during the hydride shift the C-O-1 and C-O-2 bonds of the substrate are polarized by the close approach of the site [2] cation. In the transition-state analogue this cation is observed at a site [2'], 1.0 A from site [2] and about 2.7 A from O-1 and O-2 of the substrate. It is likely that co-ordination of the cation to O-1 and O-2 would be concomitant with ionisation of the sugar hydroxyl group. The polarisation of C-O-1 and C-O-2 is assisted by the co-ordination of O-2 to cation [1] and O-1 to a lysine side-chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Observations of reaction intermediates and the mechanism of aldose-ketose interconversion by D-xylose isomerase.

Crystallographic studies of D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) incubated to equilibrium with substrate/product mixtures of xylose and xylulose show electron density for a bound intermediate. The accumulation of this bound intermediate shows that the mechanism is a non-Michaelis type. Carrell et al. [Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch, D. & Biellmann, J.-F. (1989) Proc. Natl. Acad. Sci. USA 86, 4440-4444] and the present authors studied crystals of the enzyme-substrate complex under different conditions and made different interpretations of the substrate density, leading to different conclusions about the enzyme mechanism. All authors agree that the bound intermediate of the sugar is in an open-chain form. It is suggested that the higher-temperature study of Carrell et al. may have produced an equilibrium of multiple states, whose density fits poorly to the open-chain substrate, and led to incorrect interpretation. The two groups also bound different closed-ring sugar analogues to the enzyme, but these analogues bind differently. A possible explanation consistent with all the data is that the enzyme operates by a hydride shift mechanism.

Crystallization and preliminary diffraction studies of Erythrina trypsin inhibitor.

Crystals of an inhibitor of trypsin and tissue plasminogen activator from seeds of the legume Erythrina caffra have been obtained by vapour diffusion. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22) with cell parameters 73.4 A and 143.0 A. There is one molecule in the asymmetric unit. The crystals diffract to beyond 2.5 A resolution.

Structure of tyrosyl-tRNA synthetase refined at 2.3 A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate.

The crystal structure of tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus has been refined to a crystallographic R-factor of 22.6% at 2.3 A resolution using a restrained least-squares procedure. In the final model the root-mean-square deviation from ideality for bond distances is 0.018 A and for angle distances is 0.044 A. Each monomer consists of three domains: an alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet, an alpha-helical domain (248 to 318) containing five helices, and a disordered C-terminal domain (319 to 418) for which the electron density is very weak and where it has not been possible to trace the polypeptide chain. Complexes of the enzyme with the catalytic intermediate tyrosyl adenylate and the inhibitor tyrosinyl adenylate have also been refined to R-factors of 23.9% at 2.8 A resolution and 21.0% at 2.7 A resolution, respectively. Formation of the complexes results in some crystal cracking, but there is no significant difference in the conformation of the polypeptide chain of the three structures described here. The relative orientation of the alpha/beta and alpha-helical domains is similar to that previously observed for the "A" subunit of a deletion mutant lacking the C-terminal domain. Differences between these structures are confined to surface loops that are involved in crystal packing. Tyrosyl adenylate and tyrosinyl adenylate bind in similar conformations within a deep cleft in the alpha/beta domain. The tyrosine moiety is in the equivalent position to that occupied by tyrosine in crystals of the truncated mutant and makes similar strong polar interactions with the enzyme. The alpha-phosphate group interacts with the main-chain nitrogen of Asp38. The two hydroxyl groups of the ribose form strong interactions with the protein. The 2'-hydroxyl group interacts with the carboxylate of Asp194 and the main-chain nitrogen of Gly192 while the 3'-hydroxyl interacts with a tightly bound water molecule (Wat326). The adenine moiety appears to make no significant polar interactions with the protein. The results of site-directed mutagenesis studies are examined in the light of these refined structures.