J. Habash

The recording and analysis of synchrotron X-radiation Laue diffraction photographs

Transmission Laue diffraction photographs can be recorded with short exposure times from stationary macromolecular and small-molecule crystals. With the use of a broad wavelength band a very large number of reflections is stimulated in a single `snapshot of large regions of reciprocal space. Processing software has been developed which allows quantitation of the Laue data without resort to monochromatic data. The procedures have been developed and the software strategies optimized by using test data recorded on the SRS wiggler from a protein, pea lectin, and small-molecule crystals. These latter include an organic molecule, trimethyl-1H-2,1,3-benzophosphadiazine-4(3H)-thione 2,2-disulfide, referred to as BPD, and a rhodium complex, [Rh6(CO)14(dppm)], where dppm is Ph2PCH2PPh2, referred to as RHCOP. Monochromatic data were available for comparison.

Refined structure of concanavalin A complexed with methyl α-d-mannopyranoside at 2.0 Å resolution and comparison with the saccharide-free structure

The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 A resolution, which was the first detailed study of lectin-saccharide interactions.

Active site of trypanothione reductase : a target for rational drug design

The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.

X-Ray and molecular dynamics studies of concanavalin-A glucoside and mannoside complexes Relating structure to thermodynamics of binding

Crystallographic and computational methods have been used to study the binding of two monosaccharides (glucoside and mannoside) to concanavalin-A. The 2 A structure of glucoside bound concanavalin-A is reported and compared with the 2 A structure of the mannoside complex. The interaction energies of the substrate in each crystallographic subunit were calculated by molecular mechanics and found to be essentially the same for both sugars. Further energy minimisation of the active site region of the subunits did not alter this conclusion. Information from crystallographic B-factors was interpreted in terms of mobility of the sugars in the combining site. Molecular dynamics (MD) was employed to investigate mobility of the ligands at the binding sites. Switching between different binding states was observed for mannoside over the ensemble in line with the crystallographic B-factors. A calculated average interaction energy was found to be more favourable for mannoside than glucoside, by 4.9±3.6 kcal mol-1 (comparable with the experimentally determined binding energy difference of 1.6±0.3 kcal mol-1). However, on consideration of all terms contributing to the binding enthalpy a difference is not found. This work demonstrates the difficulty in relating structure to thermodynamic properties, but suggests that dynamic models are needed to provide a more complete picture of ligand–receptor interactions.

Trends and Challenges in Experimental Macromolecular Crystallography

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 10 5 proteins that is underway, raises expectations for equivalent three dimensional structural databases.

Direct determination of the positions of the deuterium atoms of the bound water in ­concanavalin A by neutron Laue crystallography

The correct positions of the deuterium (D) atoms of many of the bound waters in the protein concanavalin A are revealed by neutron Laue diffraction. The approach includes cases where these water D atoms show enough mobility to render them invisible even to ultra-high resolution synchrotron-radiation X-ray crystallography. The positions of the bound water H atoms calculated on the basis of chemical and energetic considerations are often incorrect. The D-atom positions for the water molecules in the Mn-, Ca- and sugar-binding sites of concanavalin A are described in detail.

Unravelling the structural chemistry of the colouration mechanism in lobster shell

Biochemistry, biological crystallography, spectroscopy, solution X-ray scattering and microscopy have been applied to study the molecular basis of the colouration in lobster shell. This article presents a review of progress concentrating on recent results but set in the context of more than 50 years of work. The blue colouration of the carapace of the lobster Homarus gammarus is provided by a multimolecular carotenoprotein, alpha-crustacyanin. The complex is a 16-mer of five different subunits each binding the carotenoid, astaxanthin (AXT). A breakthrough in the structural studies came from the determination of the structure of beta-crustacyanin (protein subunits A1 with A3 with two shared bound astaxanthins). This was solved by molecular replacement using apocrustacyanin A1 as the search motif. A molecular movie has now been calculated by linear interpolation based on these two end-point protein structures, i.e. apocrustacyanin A1 and A1 associated with the two astaxanthins in beta-crustacyanin, and is presented with this paper. This movie highlights the structural changes forced upon the carotenoid on complexation. In contrast, the protein-binding site remains relatively unchanged in the binding region, but there is a large conformational change occurring in a more remote surface-loop region. It is suggested here that this loop could be important in complexation of AXT and contributes to the spectral properties. Also presented here is the first observation of single-crystal diffraction of the full alpha-crustacyanin complex comprising 16 protein subunits and 16 bound AXT molecules (i.e eight beta-crustacyanins) at 5 A resolution. Optimization of crystallization conditions is still necessary as these patterns show multiple crystallite character, however, 10 A resolution single-crystal diffraction has now been achieved. Provision of the new SRS MPW 10 and SRS MPW 14 beamline robotic systems will greatly assist in the surveying of the many alpha-crustacyanin crystallization trials that are being made. New solution X-ray scattering (SXS) measurements of beta- and alpha-crustacyanin are also presented. The beta-crustacyanin SXS data serve to show how the holo complex fits the SXS curve, whereas the apocrustacyanin A1 homodimer from the crystal data naturally does not. Reconstructions of alpha-crustacyanin were accomplished from its scattering-profile shape. The most plausible ultrastructure, based on a fourfold symmetry constraint, was found to be a stool with four legs. The latter is compared with published electron micrographs. A detailed crystal structure of alpha-crustacyanin is now sought in order to relate the full 150 nm bathochromic shift of AXT to that complete molecular structure, compared with the 100 nm achieved by the beta-crustacyanin protein dimer alone. Rare lobster colourations have been brought to attention as a result of this work and are discussed in an appendix.

Neutron Laue diffraction study of concanavalin A The proton of Asp28

Neutron Laue data collection, which harnesses a broader wavelength band emitted from the neutron source, opens up the prospect of studying larger proteins and/or using smaller protein crystals than is possible with monochromatic neutron protein crystallography data collection methods. Concanavalin A, a 25 kDa† protein was used in this study, albeit with a rather large crystal of 1.2×1.8×2.2 mm. Data in a resolution range of 8–2.75 Awere used to refine the protein structure, which included many H/D sites; the final R-factor for the protein model and 61 waters was 0.207 (Rfree = 0.310) for 4909 unique reflections. In particular, for example, the proton on Asp28 of concanavalin A, located previously by our 0.94 Asynchrotron X-ray study, was also found in this neutron study; thus the two methods confirm each other. The Asp28 proton was found not to exchange, under the deuteriation conditions used. Negative neutron density was also observed for the manganese binding site consistent with the negative neutron scattering factor for this element. Concanavalin A is one of the first proteins studied by the neutron Laue technique. The limited exchange of H for D almost certainly can be improved upon thus reducing the proton background in the diffracton pattern. This in turn would allow the weaker, high-resolution reflections, to be recorded.

Refined structure of cadmium-substituted concanavalin A at 2.0 A resolution.

The three-dimensional structure of cadmium-substituted concanavalin A has been refined using X-PLOR. The R factor on all data between 8 and 2 A is 17.1%. The protein crystallizes in space group I222 with cell dimensions a = 88.7, b = 86.5 and c = 62.5 A and has one protein subunit per asymmetric unit. The final structure contains 237 amino acids, two Cd ions, one Ca ion and 144 water molecules. One Cd ion occupies the transition-metal binding site and the second occupies an additional site, the coordinates of which were first reported by Weinzierl & Kalb [FEBS Lett. (1971), 18, 268-270]. The additional Cd ion is bound with distorted octahedral symmetry and bridges the cleft between the two monomers which form the conventional dimer of concanavalin A. This study provides a detailed analysis of the refined structure of saccharide-free concanavalin A and is the basis for comparison with saccharide complexes reported elsewhere.

Initiating a crystallographic study of trypanothione reductase

We have obtained well-ordered single crystals of the flavoenzyme trypanothione reductase from Crithidia fasciculata. The crystals are tetragonal rods with unit cell dimensions a = 128.6 A, c = 92.5 A. The diffraction pattern corresponds to a primitive lattice. Laue class 4/m. Diffraction to better than 2.4 A has been recorded at the Daresbury Synchrotron. The accurate elucidation of the three-dimensional structure of this enzyme is required to support the rational design of compounds active against a variety of tropical diseases caused by trypanosomal parasites.