A family of killer toxins. Exploring the mechanism of ADP-ribosylating toxins.

The ADP-ribosylating toxins (ADPRTs) are a family of toxins that catalyse the hydrolysis of NAD and the transfer of the ADP-ribose moiety onto a target. This family includes many notorious killers, responsible for thousands of deaths annually including: cholera, enterotoxic Escherichia coli, whooping cough, diphtheria and a plethora of Clostridial binary toxins. Despite their notoriety as pathogens, the ADPRTs have been extensively used as cellular tools to study and elucidate the functions of the small GTPases that they target. There are four classes of ADPRTs and at least one structure representative of each of these classes has been determined. They all share a common fold and several motifs around the active site that collectively facilitate the binding and transfer of the ADP-ribose moiety of NAD to their protein targets. In this review, we present an overview of the physiology and cellular qualities of the bacterial ADPRTs and take an in-depth look at the structural motifs that differentiate the different classes of bacterial ADPRTs in relation to their function.

Superantigens as immunomodulators: recent structural insights.

Superantigens interact with major histocompatibility complex (MHC) class II molecules and T-cell receptors (TcRs) forming a trimolecular complex which is able to induce proliferation and cytokine production in T cells. Although superantigens appear to act through a common mechanism, they very in many of their specific interactions and biological properties. X-ray crystallographic studies and biochemical experiments have now established that cross-linking of MHC class II molecules and the TcR by superantigens can occur in a number of different modes.

Crystal structures of guinea-pig, goat and bovine alpha-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase.

BACKGROUND: The regulation of milk lactose biosynthesis is highly dependent on the action of a specifier protein, alpha-lactalbumin (LA). Together with a glycosyltransferase, LA forms the enzyme complex lactose synthase. LA promotes the binding of glucose to the complex and facilitates the biosynthesis of lactose. To gain further insight into the molecular basis of LA function in lactose synthase we have determined the structures of three species variants of LA. RESULTS: The crystal structures of guinea-pig, goat and a recombinant from of bovine LA have been determined using molecular replacement techniques. Overall, the structures are very similar and reflect their high degree of amino acid sequence identity (66-94%). Nonetheless, the structures show that a portion of the molecule (residues 105-110), known to be important for function, exhibits a variety of distinct conformers. This region lies adjacent to two residues (Phe31 and His32) that have been implicated in monosaccharide binding by lactose synthase and its conformation has significant effects on the environments of these functional groups. The crystal structures also demonstrate that residues currently implicated in LA's modulatory properties are located in a region of the structure that has relatively high thermal parameters and is therefore probably flexible in vivo. CONCLUSIONS: LA's proposed interaction site for the catalytic component of the lactose synthase complex is primarily located in the flexible C-terminal portion of the molecule. This general observation implies that conformational adjustments may be important for the formation and function of lactose synthase.

Crystal structure of the superantigen enterotoxin C2 from Staphylococcus aureus reveals a zinc-binding site.

BACKGROUND: Staphylococcus aureus enterotoxin C2 (SEC2) belongs to a family of proteins, termed 'superantigens', that form complexes with class II MHC molecules enabling them to activate a substantial number of T cells. Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties. Comparison of the structure of SEC2 with those of two other superantigens--staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)--may provide insight into their mode of action. RESULTS: The crystal structure of SEC2 has been determined at 2.0 A resolution. The overall topology of the molecule resembles that of SEB and TSST-1, and the regions corresponding to the MHC class II and T-cell receptor binding sites on SEB are quite similar in SEC2. A unique feature of SEC2 is the presence of a zinc ion located in a solvent-exposed region at the interface between the two domains of the molecule. The zinc ion is coordinated to Asp83, His118, His122 and Asp9* (from the neighbouring molecule in the crystal lattice). Atomic absorption spectrometry demonstrates that zinc is also bound to SEC2 in solution. CONCLUSIONS: SEC2 appears to be capable of binding to MHC class II molecules in much the same manner as SEB. However, structure-function studies have suggested an alternative binding mode that involves a different site on the toxin. The zinc ion of SEC2 lies within this region and thus may be important for complex formation, for example by acting as a bridge between the two molecules. Other possible roles for the metal cation, including a catalytic one, are also considered.

Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins.

BACKGROUND: The Charcot-Leyden crystal (CLC) protein is a major autocrystallizing constituent of human eosinophils and basophils, comprising approximately 10% of the total cellular protein in these granulocytes. Identification of the distinctive hexagonal bipyramidal crystals of CLC protein in body fluids and secretions has long been considered a hallmark of eosinophil-associated allergic inflammation. Although CLC protein possesses lysophospholipase activity, its role(s) in eosinophil or basophil function or associated inflammatory responses has remained speculative. RESULTS: The crystal structure of the CLC protein has been determined at 1.8 A resolution using X-ray crystallography. The overall structural fold of CLC protein is highly similar to that of galectins -1 and -2, members of an animal lectin family formerly classified as S-type or S-Lac (soluble lactose-binding) lectins. This is the first structure of an eosinophil protein to be determined and the highest resolution structure so far determined for any member of the galectin family. CONCLUSIONS: The CLC protein structure possesses a carbohydrate-recognition domain comprising most, but not all, of the carbohydrate-binding residues that are conserved among the galectins. The protein exhibits specific (albeit weak) carbohydrate-binding activity for simple saccharides including N-acetyl-D-glucosamine and lactose. Despite CLC protein having no significant sequence or structural similarities to other lysophospholipase catalytic triad has also been identified within the CLC structure, making it a unique dual-function polypeptide. These structural findings suggest a potential intracellular and/or extracellular role(s) for the galectin-associated activities of CLC protein in eosinophil and basophil function in allergic diseases and inflammation.

Microbial superantigens: from structure to function.

Superantigens are highly potent immune stimulators with a unique ability to interact simultaneously with MHC class II molecules and T cell receptors, forming a trimolecular complex that induces profound T-cell proliferation and massive cytokine production. Recent structural studies have provided a wealth of information regarding these complex interactions, and it is now emerging that, despite their common 3-D architecture, superantigens are able to crosslink MHC class II molecules and T cell receptors in a variety of ways.

Crystal structure of microbial superantigen staphylococcal enterotoxin B at 1.5 A resolution: implications for superantigen recognition by MHC class II molecules and T-cell receptors.

Staphylococcal enterotoxin B is a member of a family of toxins known as superantigens that activate a large number of T-cells (up to 20%) by cross-linking MHC class II molecules with T-cell receptors in a Vbeta-restricted fashion. The crystal structure of staphylococcal enterotoxin B presented here has been determined at 1.5 A resolution, the highest resolution so far for a superantigen. The final model contains 1948 protein atoms and 177 water molecules and has excellent geometry with root-mean-square (rms) deviation of 0.007 A and 1.73 degrees in bond lengths and bond angles, respectively. The overall fold is similar to that of other microbial superantigens, but as it lacks the zinc-binding site found in other members of this family, such as staphylococcal enterotoxin A, C2 and D, this enterotoxin possesses only one MHC class II binding site. Comparison of the crystal structure of free SEB and in complex with an MHC class II molecule revealed no major changes in the MHC-binding site upon complex formation. However, a number of water molecules found in the free SEB may be displaced in the complex or contribute further to its stability. Detailed analysis of the TcR-binding site of SEB, SEA and SEC2 shows significant differences which may account for the ability of each superantigen to bind specific Vbeta sequences.

Crystal structure of human alpha-lactalbumin at 1.7 A resolution.

The three-dimensional X-ray structure of human alpha-lactalbumin, an important component of milk, has been determined at 1.7 A (0.17 nm) resolution by the method of molecular replacement, using the refined structure of baboon alpha-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2(1)2(1)2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F greater than or equal to 2 sigma (F)) between 8 and 1.7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9 degrees for bond angles. Superposition of the human and baboon alpha-lactalbumin structures yields a root-mean-square difference of 0.67 A for the 123 structurally equivalent C alpha atoms. The C terminus is flexible in the human alpha-lactalbumin molecule. The striking structural resemblance between alpha-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.

Crystallization and preliminary X-ray diffraction analysis of foot-and-mouth disease virus.

Foot-and-mouth disease virus has been crystallized with the objectives of (1) determining the composition and conformation of the major immunogenic site(s) and (2) comparing its structure with those of the related polio, rhino and Mengo viruses, representing the other three genera of the picornaviruses. Most of the work has been done with virus strain O1BFS 1860, which crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. Virus recovered from crystals was infectious, and was indistinguishable from native virus both in protein composition and buoyant density. The stability of the crystals in the X-ray beam was comparable with that of other picornavirus crystals and they diffracted to a resolution of better than 2.3 A. Initial analysis of the X-ray diffraction data shows the virus to be positioned on a point of 23 symmetry in a close-packed array so that examples of all the icosahedral symmetry elements, except the 5-fold axes, are expressed crystallographically. The cell dimensions are a = b = c = 345 A, alpha = beta = gamma = 90 degrees, with a space group of I23. The diameter of the virus particle is 300 A. Despite the small size of the crystals, diffraction data have been collected to a reasonable resolution using a synchrotron source. Phasing of the diffraction data will be attempted using the methods of molecular replacement.

Crystallographic binding studies on the allosteric inhibitor glucose-6-phosphate to T state glycogen phosphorylase b.

Glucose-6-phosphate is an important allosteric inhibitor of glycogen phosphorylase b that restrains the enzyme in the inactive state in resting muscle. A crystallographic binding study by diffusion of glucose-6-phosphate into performed crystals of T state phosphorylase b has been carried out at 2.3 A resolution and the structure refined by restrained crystallographic least-squares and simulated annealing to give a crystallographic R-value of 0.203. The inhibitor binds at the AMP allosteric effector site at the subunit-subunit interface of the dimer. The phosphate groups of the glucose-6-phosphate and AMP occupy partially overlapping sites and make similar contacts to two arginine residues (Arg309 and Arg310) but in glucose-6-phosphate there is a contact to a third arginine (Arg242). The glucopyranose of glucose-6-phosphate and the adenine ribose of AMP occupy different positions. Including the contacts to the three arginine residues by the phosphate group, the glucose-6-phosphate makes a total of 11 hydrogen-bonds to the enzyme and all but one of these are to charged groups. The O-2 hydroxyl hydrogen-bonds to the main-chain carbonyl oxygen of Val40' from the other subunit and this interaction appears important for the allosteric response. There are substantial conformational changes both in the vicinity of the glucose-6-phosphate (involving for example Phe196 and Arg309) and at the subunit interface (involving residues 42' to 51' and 192 to 196). These shifts tighten the binding of the inhibitor and the interface. Comparison of the glucose-6-phosphate complex with the T state native phosphorylase b and the R state phosphorylase a structures shows that there is a graded response from T state glucose-6-phosphate complex through T state phosphorylase b to R state phosphorylase a that suggests that glucose-6-phosphate promotes a tight structure that is more "tensed" than native T state phosphorylase b. The results show how the same allosteric effector site can exhibit a tight binding site for the activator AMP in the R state structure and a tight binding site for glucose-6-phosphate in the modified T state structure.

Crystallization and preliminary X-ray analysis of human angiogenin.

Crystals of recombinant human angiogenin have been grown from solutions containing sodium potassium tartrate and polyethylene glycol as precipitants. They belong to the space group C222(1) (a = 83.36 A, b = 120.64 A, c = 37.72 A) and contain a single molecule in the asymmetric unit. The crystals diffract to at least 2.3 A resolution and are suitable for three-dimensional X-ray structural analysis.

Purification, crystallization and preliminary X-ray analysis of toxic shock syndrome toxin-1 from Staphylococcus aureus.

High yields of toxic shock syndrome toxin-1, from Staphylococcus aureus, have been purified (> 99%) using a novel, simple, two-step procedure involving dye ligand chromatography. Crystals suitable for X-ray diffraction work were obtained by vapour diffusion using ammonium sulphate and polyethylene glycol as precipitants. They belong to the orthorhombic space group C222(1), with unit cell dimensions a = 108.6 A, b = 177.6 A and c = 97.5 A, with three molecules per asymmetric unit. The crystals diffract to at least 2.5 A resolution and are suitable for three-dimensional X-ray structural analysis.

Crystallization and preliminary X-ray analysis of a microbial superantigen staphylococcal enterotoxin C2.

High yields of staphylococcal enterotoxin C2, from Staphylococcus aureus, have been purified using dye ligand chromatography. Crystals suitable for X-ray diffraction work were obtained by vapour diffusion using ammonium sulphate and polyethylene glycol as precipitants. They belong to the tetragonal space group P4(1)22 with unit cell dimensions a = b = 43.2 A and c = 290.9 A with one molecule per asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for three-dimensional X-ray structural analysis.

The binding of 2-deoxy-D-glucose 6-phosphate to glycogen phosphorylase b: kinetic and crystallographic studies.

Kinetic and crystallographic studies have characterized the effect of 2-deoxy-glucose 6-phosphate on the catalytic and structural properties of glycogen phosphorylase b. Previous work on the binding of glucose 6-phosphate, a potent physiological inhibitor of the enzyme, to T state phosphorylase b in the crystal showed that the inhibitor binds at the allosteric site and induces substantial conformational changes that affect the subunit-subunit interface. The hydrogen-bond from the O-2 hydroxyl of glucose 6-phosphate to the main-chain oxygen of Val40' represents the only hydrogen bond from the sugar to the other subunit, and this interaction appears important for promoting a more "tensed" structure than native T state phosphorylase b. 2-Deoxy-glucose 6-phosphate acts competitively with both the activator AMP and the substrate glucose 1-phosphate, with Ki values of 0.53 mM and 1.23 mM, respectively. The binding of 2-deoxy-glucose 6-phosphate to T state glycogen phosphorylase b in the crystal, has been investigated and the complex phosphorylase b: 2-deoxy-glucose 6-phosphate has been refined to give a crystallographic R factor of 17.3%, for data between 8 A and 2.3 A. 2-Deoxy-glucose 6-phosphate binds at the allosteric site as the a anomer and adopts a different conformation compared to glucose 6-phosphate. The two conformations differ by 160 degrees in the torsion angle about the C-5-C-6 bond. The contacts from the phosphate group are essentially identical to those made by the phosphate of glucose 6-phosphate but the 2-deoxy glucosyl moiety binds in a quite different orientation compared to the glucosyl of glucose 6-phosphate. 2-Deoxy-glucose 6-phosphate can be accommodated in the allosteric site with very little change in the protein, while structural comparisons show that the phosphorylase b: 2-deoxy-glucose 6-phosphate complex structure is overall more similar to a glucose-like complex than to the Glc-6-P complex structure.

A structural and functional comparison of staphylococcal enterotoxins A and C2 reveals remarkable similarity and dissimilarity.

Staphylococcal enterotoxins and toxic shock syndrome toxin-1 are known as superantigens due to their ability to activate a large number of T-cells by crosslinking the major histocompatibility complex class II molecules with the T-cell receptor. Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties. A structural comparison of staphylococcal enterotoxins A and C2, members of the staphylococcal superantigens, has shown large conformational differences at the putative TcR interaction site (loops between alphaN-alpha2, alpha4-beta9 and beta10-alpha5 in staphylococcal enterotoxin A) that could explain the variability in their T-cell receptor specificity. A common Zn2(+)-binding site was identified in both staphylococcal enterotoxin A and C2 that is superimposable but differs somewhat in its coordination geometry between the two molecules.

The refined crystal structure of toxic shock syndrome toxin-1 at 2.07 A resolution.

The pyrogenic toxin toxic shock syndrome toxin-1 from Staphylococcus aureus is a causative agent of the toxic shock syndrome disease. It belongs to a family of proteins known as superantigens that cross-link major histocompatibility class II molecules and T-cell receptors leading to the activation of a substantial number of T cells. The crystal structure of this protein has been refined to 2.07 A with an Rcryst value of 20.4% for 51,240 reflections. The final model contains three molecules in the asymmetric unit with good stereochemistry and a root-mean-square deviation of 0.009 A and 1.63 from ideality for bond lengths and bond angles, respectively. The overall fold is considerably similar to that of other known microbial superantigens (staphylococcal enterotoxins). However, a detailed structural analysis shows that toxic shock syndrome toxin-1 lacks several structural features that affect its specificity for V beta elements of the T-cell receptor and also its recognition by major histocompatibility class II molecules.

Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth.

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.

Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme.

The solution of the structure of alpha-lactalbumin from baboon milk (Papio cynocephalus) at 4.5 A resolution using the isomorphous replacement method has been reported previously. Initial refinement on the basis of these low-resolution studies was not successful because of the poor isomorphism of the best heavy-atom derivative. Because of the striking similarity between the structure of lysozyme and alpha-lactalbumin, a more cautious molecular replacement approach was tried to refine the model. Using hen egg-white lysozyme as the starting model, preliminary refinement was performed using heavily constrained least-squares minimization in reciprocal space. The model was further refined using stereochemical restraints at 1.7 A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A, and for angle distances it is 0.027 A. The refinement was carried out using the human alpha-lactalbumin sequence and "omit maps" calculated during the course of refinement indicated eight possible sequence changes in the baboon alpha-lactalbumin X-ray sequence. During the refinement, a tightly bound calcium ion and 150 water molecules, of which four are internal, have been located. Some of the water molecules were modelled for disordered side-chains. The co-ordination around the calcium is a slightly distorted pentagonal bipyramid. The Ca-O distances vary from 2.2 A to 2.6 A, representing a tight calcium-binding loop in the structure. The calcium-binding fold only superficially resembles the "EF-hand" and presumably has no evolutionary relationship with other EF-hand structures. The overall structure of alpha-lactalbumin is very similar to that of lysozyme. All large deviations occur in the loops where all sequence deletions and insertions are found. The C terminus appears to be rather flexible in alpha-lactalbumin compared to lysozyme. The experimental evidence supports the earlier predictions for the alpha-lactalbumin structure that were based upon the assumption that alpha-lactalbumin and lysozyme have similar three-dimensional structures, with minimal deletions and insertions. A detailed comparison of the two structures shows striking features as well as throwing some light on the evolution of these two proteins from a common precursor.

Refined crystal structure of the phosphorylase-heptulose 2-phosphate-oligosaccharide-AMP complex.

The crystal structure of phosphorylase b-heptulose 2-phosphate complex with oligosaccharide and AMP bound has been refined by molecular dynamics and crystallographic least-squares with the program XPLOR. Shifts in atomic positions of up to 4 A from the native enzyme structure were correctly determined by the program without manual intervention. The final crystallographic R value for data between 8 and 2.86 A resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 A for all protein atoms. The results confirm the previous observation that there is a direct hydrogen bond between the phosphate of heptulose 2-phosphate and the pyridoxal phosphate 5'-phosphate group. The close proximity of the two phosphates is stabilized by an arginine residue, Arg569, which shifts from a site buried in the protein to a position where it can make contact with the product phosphate. There is a mutual interchange in position between the arginine and an acidic group, Asp283. These movements represent the first stage of the allosteric response which converts the catalytic site from a low to a high-affinity binding site. Communication of these changes to other sites is prevented in the crystal by the lattice forces, which also form the subunit interface. The constellation of groups in the phosphorylase transition state analogue complex provides a structural basis for understanding the catalytic mechanism in which the cofactor pyridoxal phosphate 5'-phosphate group functions as a general acid to promote attack by the substrate phosphate on the glycosidic bond when the reaction proceeds in the direction of glycogen degradation. In the direction of glycogen synthesis, stereoelectronic effects contribute to the cleavage of the C-1-O-1 bond. In both reactions the substrate phosphate plays a key role in transition state stabilization. The details of the oligosaccharide, maltoheptaose, interactions with the enzyme at the glycogen storage site are also described.

A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.