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Six different crystal forms of the long neurotoxin α-cobratoxin isolated from the venom of Naja naja siamensis have been obtained and the conditions for their crystallisation are described. Heavy atom derivatives for two of these forms have been prepared and have proven suitable for high-resolution X-ray studies and, with one of them, a 0.28-nm resoution structure has been determined. All but one of the crystal forms were grown between pH 2 and pH 3. Two novel suggestions are made which may be applicable to other protein crystallisation studies. One involves cocrystallisation with mercurous iodide and the other describes a two-step microdialysis procedure. first against salt solution, followed by analysis against poly(ethylene glycol). [ABSTRACT FROM AUTHOR]

The complex of fatty-acid-binding protein (FABP) from bovine heart (cFABP, pI 4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle-shaped crystals belong to the monoclinic space group C2, with unit-cell constants a= 5.262(6)nm, b = 7.631(8)nm, c = 3.945(5)nm and β = 94.47(9)°. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods. An initial model for the three-dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T. A., Bergfors, T., Sedzik, J. & Unge, T. (1988) EMBO J. 7, 1597–1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all-atom and a united-atom force field. The optimized models were used to determine the crystal structure of cFABP by molecular-replacement techniques. The structure was refined by simulated annealing to R = 0.267. As the bound lipid is heterogeneous, it could not be located in the electron-density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel β strands forming a β barrel, and by two α helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters. [ABSTRACT FROM AUTHOR]

Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase T1, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted. The mutants are His40 → Thr, Glu58 → Asp, Glu58 → Gln, His92 → Ala and His92 → Phe. His92 → Ala and His92 → Phe mutants are inactive. On the basis of their kcat/Km values, the mutants Glu58 → Asp and Glu58 → Gln show 10% and 7% residual activity, relative to wild-type RNase T1, whereas the His40 → Thr mutant shows 2% activity. The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three-dimensional structure of the enzyme. [ABSTRACT FROM AUTHOR]

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39-His69-Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34-Cys123 and Cys178-Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel β-sheet decorated by six α-helices, three short antiparallel β-sheets, 18 β-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132-Gly136), probably associated with a cis→trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132-Leu133-Gly134 on one side and Gly100-Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39-His69-Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent. [ABSTRACT FROM AUTHOR]

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53%(v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2′-guanylic acid (2′GMP) and 0.02% NaN3 (pH 6.2–7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01–0.05 M sodium acetate, 0.25–0.5% 2′GMP, 0.02% NaN3 and 2–5 nM calcium acetate (pH 4.0–4.4) in the presence of 50–75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and a = 4.44nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2′-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2′GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 · 2′GMP complex is under way. [ABSTRACT FROM AUTHOR]

Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents. Crystals have tetragonal space group P42212, a = b = 11.27 nm, c = 20.14 nm, and contain one quarter molecule per asymmetric unit. X-rays are diffracted to 0.35-rim resolution on still photographs. Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X-rays. Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase. [ABSTRACT FROM AUTHOR]

The molecular structure of the uridine analog 6,7-dimethyllumazine-N-1-β-D.ribofuranoside has been determined from 4114 three-dimensional X-ray data. The substance crystallized in the monoclinic space group P21 with two crystallographically independent molecules per asymmetric unit. The intensity data were measured diffractometrically, and the positions of the 46 nonhydrogen atoms were located by direct methods. The atomic parameters were refined by fullmatrix least-squares methods to a discrepancy index of 5.9%. Both molecules are in the unusual syn conformation with C(2')-endo sugar puckering and a gauche, gauche conformation about the C(')-C(5') bond; thus an intramolecular O(5')-H · O(2) hydrogen bond can be formed. The molecules are packed such that the "hydrophobic" pteridine heterocycles are stacked in endless ribbons which are separated by regions containing the sugar residues. The lumazine substituents at O(2), 0(4) and N(3)-H but not N(5) and N(8) are involved in hydrogen bonding. A discussion of the possible biochemical consequences are given. [ABSTRACT FROM AUTHOR]

Hen egg-white avidin was crystallized at pH 6 from solutions containing 8-9% (w/v) poly(ethyleneglycol) 6000 as precipitant. X-ray investigation showed that the crystalsare orthorhombic with cell constants a = 7.47 nm, b = 8.00 nm, c = 4.31 nm and belong to space group P212121. They contain half of a tetrameric molecule in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 Å resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1-MAGE-A1. Fab-Hyb3 employs a diagonal docking mode resembling that of T cell receptors. However, other than these natural ligands, the antibody uses only four of its six complementarity-determining regions for direct interactions with the target. It recognizes the C-terminal half of the MAGE-A1 peptide, the HLA-A1 α1-helix, and N-terminal residues of the α2-heIix, accompanied by a large tilting angle between the two types of molecules within the complex. Interestingly, only a single hydrogen bond between a peptide side chain and Fab·Hyb3 contributes to the interaction, but large buried surface areas with pronounced shape complementarity assure high affinity and specificity for MAGE-A1. The HLA-A1·MAGE-A1·antibody structure is discussed in comparison with those of natural ligands recognizing HLA·peptide complexes. [ABSTRACT FROM AUTHOR]

Molecular mimicry is discussed as a possible mechanism that may contribute to the development of autoimmune diseases. It could also be involved in the differential association of the human major histocompatibility subtypes HLA-B*2705 and HLA-B*2709 with ankylosing spondylitis. These two subtypes differ only in residue 116 of the heavy chain (Asp in B*2705 and His in B*2709), but the reason for the differential disease association is not understood. Using x-ray crystallography, we show here that the viral peptide pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236244) of Epstein-Barr virus) is presented by the B*2705 and B*2709 molecules in two drastically deviating conformations. Extensive structural similarity between pLMP2 and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400-408)) is observed only when the peptides are presented by B*2705 because of a salt bridge between Arg5 of both peptides and the subtype-specific heavy chain residue Asp116. Combined with functional studies using pLMP2/pVIPR-cross-reactive cytotoxic T cell lines and clones, together with target cells presentlng these peptides or a modified peptide analogue, our results reveal that a pathogen-derived peptide can exhibit major histocompatibility complex class I subtypedependent, drastically distinct binding modes. Furthermore, the results demonstrate that molecular mimicry between pLMP2 and pVIPR in the HLA-B27 context is an allele-dependent property. [ABSTRACT FROM AUTHOR]