David S. Moss

PROCHECK: a program to check the stereochemical quality of protein structures

The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.

X-ray analysis of the eye lens protein γ-II crystallin at 1·9 Å resolution*

We report the X-ray structure analysis and refinement at 1·9 A resolution of calf γ-II crystallin, a lens-specific protein. The sequence of Croft (1972) has been modified to give a polypeptide chain of 174 residues (cf. 165). The protein has a symmetrical, hierarchical structure of two globular domains each comprising two similar “Greek key” motifs, consecutive along the polypeptide chain, and related by a pseudo 2-fold axis. The two domains pack together with a single connection and are related by a further pseudo 2-fold axis which bisects the angle between the intra-domain dyads. Forty-two pairs of Cα positions for the two most similar motifs have root-mean-square separation at best fit of 0·69 A. The N and C-terminal domains gave root-mean-square separation of 0·89 A for 82 pairs of Cα atoms at best fit. In each domain the two Greek key motifs form a pair of four-stranded antiparallel β-pleated sheets, each sheet composed of three stands from one motif and one from the other. The sheets pack together in a wedge shape, closed at the top by the loops connecting the third and fourth strands of each motif. The two strands of each motif form an extended β-hairpin which is folded on to the β-sheet. The packing of each motif into the globular domains involves a staggered bilayer of side-chains between each pair of β-sheets which does not preserve the pseudo 2-fold axes observed in the Cα position topology. In the core of each domain there are interactions between polarizable aromatic groups and sulphur-containing residues which may contribute to stability and may also serve to protect aromatic side-chains from ultraviolet light damage in the lens. At the surface of the molecule over half the ionic side-chains are closely paired, which probably stabilizes the tertiary fold and may reduce the water bound. Crystal lattice interactions are described which may be similar to those occurring in vivo in the lens between crystallins. Seven cysteine residues have been identified in the structure and these may have a role in the thermodynamic stability of the molecule, its intermolecular interactions under the normal reducing conditions of the lens, and also in the aggregation and cross-linking which occur in some forms of cataract. Three of these residues, Cys18, Cys23 and Cys74, form a cluster in the N-terminal domain. The high-resolution data from relatively aged crystals suggest that a disulphide bond exists between Cys18 and Cys23 under appropriate oxidizing conditions. Cys15 is very exposed, is involved in a crystal lattice interaction with arginine, and could form an intermolecular disulphide in solution when oxidized.

Structure of the key toxin in gas gangrene

Clostridium perfringens α-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with α-toxin. Finally, a possible mechanism for α-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

Clostridium Perfringens Epsilon-Toxin Shows Structural Similarity to the Pore-Forming Toxin Aerolysin

ε-Toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of ε-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.

TLSANL: TLS parameter-analysis program for segmented anisotropic refinement of macromolecular structures

The atomic displacements of many of the atoms in a macromolecular structure can be modelled in terms of group motions described in the harmonic approximation by T, L and S tensors. Relevant groups may be planar side groups of protein chains, units of secondary structure such as α-helices or whole protein domains. For the TLS parameters to be interpreted, they must be related to the axes of inertia of the rigid groups and, in the case of the T and S tensors, must be calculated with respect to the centre of reaction of the rigid group. A program (TLSANL) is described that analyses these 21 TLS rigid-body displacement parameters and their relation with the principal axes of the rigid body, from the output of the segmented anisotropic refinement of a macromolecular structure, as produced by a program such as RESTRAIN [Haneef, Moss, Stanford & Borkakoti (1985). Acta Cryst. A41, 426–433; Driessen, Haneef, Harris, Howlin, Khan & Moss (1989). J. Appl. Cryst. 22, 510–516].

Benchmarking pKa prediction

BackgroundpKa values are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pKa shift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data.ResultsHere we use a large dataset of experimentally-determined pKas to analyse the performance of different prediction techniques. Our work provides a benchmark of available software implementations: MCCE, MEAD, PROPKA and UHBD. Combinatorial and regression analysis is also used in an attempt to find a consensus approach towards pKa prediction. The tendency of individual programs to over- or underpredict the pKa value is related to the underlying methodology of the individual programs.ConclusionOverall, PROPKA is more accurate than the other three programs. Key to developing accurate predictive software will be a complete sampling of conformations accessible to protein structures.

Error Estimates of Protein Structure Coordinates and Deviations from Standard Geometry by Full-Matrix Refinement of γB- and βB2-Crystallin

Faster workstations with larger memories are making error estimation from full-matrix least-squares refinement a more practicable technique in protein crystallography. Using minimum variance weighting, estimated standard deviations of atomic positions have been calculated for two eye lens proteins from the inverse of a least-squares normal matrix which was full with respect to the coordinate parameters. γB-crystallin, refined at 1.49 A yielded average errors in atomic positions which ranged from 0.05 A for main-chain atoms to 0.27 A for unrestrained water molecules. The second structure used in this work was that of βB2-crystallin refined at 2.1 A resolution where the corresponding average errors were 0.08 and 0.35 A, respectively. The relative errors in atomic positions are dependent on the number and kinds of restraints used in the refinements. It is also shown that minimum variance weighting leads to mean-square deviations from target geometry in the refined structures which are smaller than the variances used in the distance weighting.

Segmented anisotropic refinement of bovine ribonuclease A by the application of the rigid-body TLS model.

The anisotropic displacements of selected rigid groups in bovine ribonuclease A have been refined from X-ray diffraction data by the application of the rigid-body TLS model. The rigid groups chosen were the side chains of tyrosine, histidine and phenylalanine and the planar side chains of aspartic acid, glutamic acid, glutamine, asparagine and arginine. The method has also been applied to the co-crystallizing active-site sulfate anion. This has enabled the description of the motion of the above-mentioned side-chain atoms by anisotropic displacement ellipsoids from a 1.45 A refinement. The hydrophobic side groups in the protein core show mainly translational motion, with mean-square librations of 20 deg2 which are similar to those found in some close-packed crystals of small organic molecules. Librational displacements are much more significant in the hydrophilic side groups where their magnitudes can be correlated with solvent accessibility. Large librations of some solvent exposed side chains correspond with the breakdown of a simple TLS model and the existence of multiple orientations of the side groups. The TLS model has also been applied to the whole protein molecule and shows that the average motion is approximately isotropic with little librational character.

Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins.

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens

Background: Clostridium perfringens toxin NetB is a key factor in avian necrotic enteritis. Results: NetB forms heptameric pores structurally similar to Staphylococcus aureus toxins but lacks a phosphocholine binding pocket. NetB activity is enhanced by cholesterol. Conclusion: NetB has distinct binding specificity, and cholesterol may act as a receptor. Significance: The structure of NetB will facilitate development of control measures against necrotic enteritis. NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.