Refinement of the crystal structure of wheat germ agglutinin isolectin 2 at 1.8 A resolution

The crystal structure of wheat germ agglutinin isolectin 2 has been refined by the restrained least-squares method of Hendrickson & Konnert (1980). The asymmetric unit of the C2 crystals contains two chemically identical promoters related by a non-crystallographic 2-fold screw operation. A total of 2290 protein atoms and 186 ordered water sites refined to a final R-factor of 0.179 and an average B-value of 21.6 A2, using 54% (15,601) of the total possible number of reflections in the resolution range 8 to 1.8 A with Fo greater than 3 sigma (Fo). The final model conforms to stereochemically correct bond distances and angles with root-mean-square (r.m.s.) values of 0.018 A and 3.3 degrees, respectively. Accuracy of this model is estimated to be 0.20 A on the basis of a Luzzati plot. Main-chain atomic positions in the two independent promoters, designated I and II, agree with an r.m.s. deviation of 0.30 A (0.58 A for all atoms), indicating identical backbone conformation. The largest discrepancies are seen at flexible surface residues. One error was detected in the amino acid sequence at position 41 (Ser), which refined satisfactorily as a Trp. Loss of electron density for residue A171 during the course of refinement suggests either disorder or absence of this C-terminal residue. The conformation of the polypeptide chain, which is folded into four homologous 43-residue domains (A, B, C and D), was analyzed in terms of dihedral angles, backbone hydrogen bond lengths and CA-atom positions. The four domains were found to be very similar according to all these criteria and superposition of their CA-atoms yielded r.m.s. distances ranging from 0.36 to 0.72 A for the six possible comparisons [corrected]. Large deviations (greater than 1.0 A) are only seen in the five-residue segments that link adjacent domains and at the N and C termini. Refinement has also allowed critical examination of each of the two unique sugar binding sites, referred to as "primary" and "secondary" sites, in different lattice environments. While the essential tyrosyl side-chain in each of these sites (Y73, Y159) assumes precise orientation for optimum hydrophobic contact with the N-acetyl methyl group of the sugar ligand, side-chains involved in hydrogen bonds (S62, E115; and S148, D29) were found to be relatively flexible and able to adapt their conformation to changes in environment. Ordered water structure present in these binding sites is not completely analogous in the different environments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histidine determination in wheat germ agglutinin isolectin by X-ray diffraction analysis

Electron density maps based on 2·4 Å and 2·2 Å X-ray diffraction data for crystals of two isolectins of wheat germ agglutinin (designated isolectins 1 and 2) were compared in terms of side-chain identities. While the primary structure of wheat germ agglutinin is not available, a partial amino acid sequence for isolectin 2 has been deduced by inspection of the electron density map and through model building. The positions of the two histidines predicted from amino acid composition studies to be present in isolectin 2 but not in isolectin 1, were located by difference Fourier techniques and analysis of the heavy-atom binding properties of these two isolectins. Both histidines were found to reside in the B-domain of the multi-domain wheat germ agglutinin protomer (A, B, C, D). Histidine 57 lies in the contact region between the two subunits near the molecular dimer axis. The side-chain of histidine 64 forms part of the primary saccharide binding site at the interface where B and C-domains of opposite protomers make contact. In addition, this histidine serves as a major target for heavy-atom binding by platinum and mercury compounds.     

Primary structure of wheat germ agglutinin isolectin 2. Peptide order deduced from X-ray structure

The complete amino acid sequence of wheat germ agglutinin isolectin 2 has been determined by the method of sequential Edman degradation and with the aid of the three-dimensional structure known from X-ray crystallography. Peptides ranging from 2 to 18 residues in length were obtained by thermolysin digestion of the S-carboxymethylated protein and purified by gel filtration and high-performance liquid chromatography. The peptide order was established primarily by matching (carboxymethyl)cysteines with the clearly defined half-cystine positions in the X-ray structure, thereby satisfying the disulfide repeat pattern observed in all four isostructural domains (A, B, C, and D) of wheat germ agglutinin, and by examination of amino acid compositions and terminal sequences of ten tryptic peptides. The unique assignment of peptides to these domains was consistent with all invariant half-cystines and glycines, as well as the single tryptophan, the two closely spaced histidines, and a number of other residues clearly identified in the X-ray structure analysis. Discrepancies between the chemical and X-ray sequences lie exclusively in poorly defined regions of the electron density map, at the N- and C-termini, and at the first intercystine loop of each domain. The latter loop was found to be eight instead of six residues in length, thus extending the size of domains A, B, and C from 41 to 43 residues and that of domain D to 42 residues. Regions of extensive interdomain homology, in addition to that of the half-cystines, are clustered at the central portion of each domain fold and are likely to be important for the integrity of the three-dimensional structure of the dimer molecule.     

Structure of calmodulin refined at 2.2 A resolution

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.     

Subunit structure of wheat germ agglutinin

Cells isolated by enzymic digestion of embryonic tendon were incubated under N₂ so that they synthesized and accumulated the unhydroxylated form of procollagen which is known as protocollagen and which is largely comprised of pro-α chains linked by interchain disulfide bonds. The cells were then exposed to O₂ so that the intracellular protocollagen was hydroxylated and secreted as procollagen. When the hydroxylation was allowed to proceed at 31° or 34°, the procollagen secreted into the medium was triple-helical but its hydroxyproline content was less than two-thirds and its hydroxylysine content was less than half the control. Even when the hydroxylation was allowed to occur at 37°, the procollagen secreted by the cells was under-hydroxylated by about 15% in terms of its hydroxyproline content and about 45% in terms of its hydroxylysine content. The results may have consequences for collagen synthesis by tendons and similar tissues $\text{in vivo}$, since temporary anoxia in such tissues may well lead to the synthesis of a less stable procollagen or to fibers of decreased tensile strength.     

Calmodulin structure refined at 1.7 A resolution.

We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 A resolution. The structure was determined by molecular replacement, using the 2.2 A published native bovine brain structure as the starting model. The final crystallographic R-factor, using 14,469 reflections in the 10.0 to 1.7 A range with structure factors exceeding 0.5 sigma, is 0.216. Bond lengths and bond angle distances have root-mean-square deviations from ideal values of 0.009 A and 0.032 A, respectively. The final model consists of 1279 non-hydrogen atoms, including four calcium ions, 1130 protein atoms, including three Asp118 side-chain atoms in double conformation, 139 water molecules and one ethanol molecule. The electron densities for residues 1 to 4 and 148 of calmodulin are poorly defined, and not included in our model, except for main-chain atoms of residue 4. The calmodulin structure from our crystals is very similar to the earlier 2.2 A structure described by Babu and coworkers with a root-mean-square deviation of 0.36 A. Calmodulin remains a dumb-bell-shaped molecule, with similar lobes and connected by a central alpha-helix. Each lobe contains three alpha-helices and two Ca2+ binding EF hand loops, with a short antiparallel beta-sheet between adjacent EF hand loops and one non-EF hand loop. There are some differences in the structure of the central helix. The crystal packing is extensively studied, and facile crystal growth along the z-axis of the triclinic crystals is explained. Herein, we describe hydrogen bonding in the various secondary structure elements and hydration of calmodulin.     

Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.     

The refined crystal structure of ribonuclease A at 2.0 A resolution

This paper describes the structure of bovine pancreatic ribonuclease A, refined by a restrained parameter least squares procedure at 2.0 A resolution, and rebuilt using computer graphics. The final agreement factor (formula see text) is 0.159. The positions of the 951 main chain atoms have been determined with an estimated accuracy of 0.17 A. In addition, the model includes a phosphate group in the active site and 176 waters, many of them with partial occupancy. The bond lengths in the refined structure of RNase A differ from the ideal values by an overall root mean square deviation of 0.022 A; the corresponding value for angle distances is 0.06 A. The root mean square deviation of planar atoms from ideality is 0.017 A, and root mean square deviation of the peptide torsion angles from 180 degrees is 3.4 degrees. The model is in good agreement with the final difference Fourier maps. Two active site histidines, His 12 and His 119, form hydrogen bonds to the phosphate ion. His 119 is also hydrogen bonded to the carboxyl of ASp 121 and His 12 to the carbonyl of Thr 45. The structure of the RNase A is very similar to that of RNase S, particularly in the active site region. The root mean square discrepancy of all atoms from residues 1 to 16 and 24 to 123 is 1.06 A and the root mean square discrepancy for the active site region is 0.6 A.     

Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution

The crystal structure of calmodulin (Mr 16,700, 148 residues) from Drosophila melanogaster as expressed in a bacterial system has been determined and refined at 2.2-A resolution. Starting with the structure of mammalian calmodulin, we produced an extensively refitted and refined model with a conventional crystallographic R value of 0.197 for the 5,239 reflections (F greater than or equal to 2 sigma (F)) within the 10.0-2.2-A resolution range. The model includes 1,164 protein atoms, 4 calcium ions, and 78 water molecules and has root mean square deviations from standard values of 0.018 A for bond lengths and 0.043 A for angle distances. The overall structure is similar to mammalian calmodulin, with a seven-turn central helix connecting the two calcium-binding domains. The "dumb-bell" shaped molecule contains seven alpha-helices and four "EF hand" calcium-binding sites. Although the amino acid sequences of mammalian and Drosophila calmodulins differ by only three conservative amino acid changes, the refined model reveals a number of significant differences between the two structures. Superimposition of the structures yields a root mean square deviation of 1.22 A for the 1,120 equivalent atoms. The calcium-binding domains have a root mean square deviation of 0.85 A for the 353 equivalent atoms. There are also differences in the amino terminus, the bend of the central alpha-helix, and the orientations of some of the side chains.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

The crystal structure of staphylococcal nuclease refined at 1.7 A resolution

The crystal structure of staphylococcal nuclease has been determined to 1.7 A resolution with a final R-factor of 16.2% using stereochemically restrained Hendrickson-Konnert least-squares refinement. The structure reveals a number of conformational changes relative to the structure of the ternary complex of staphylococcal nuclease 1,2 bound with deoxythymidine-3',5'-diphosphate and Ca2+. Tyr-113 and Tyr-115, which pack against the nucleotide base in the nuclease complex, are rotated outward creating a more open binding pocket in the absence of nucleotide. The side chains of Ca2+ ligands Asp-21 and Asp-40 shift as does Glu-43, the proposed general base in the hydrolysis of the 5'-phosphodiester bond. The significance of some changes in the catalytic site is uncertain due to the intrusion of a symmetry related Lys-70 side chain which hydrogen bonds to both Asp-21 and Glu-43. The position of a flexible loop centered around residue 50 is altered, most likely due to conformational changes propagated from the Ca2+ site. The side chains of Arg-35, Lys-84, Tyr-85, and Arg-87, which hydrogen bond to the 3'- and 5'-phosphates of the nucleotide in the nuclease complex, are unchanged in conformation, with packing interactions with adjacent protein side chains sufficient to fix the geometry in the absence of ligand. The nuclease structure presented here, in combination with the stereochemically restrained refinement of the nuclease complex structure at 1.65 A, provides a wealth of structural information for the increasing number of studies using staphylococcal nuclease as a model system of protein structure and function.     

The refined crystal structure of subtilisin Carlsberg at 2.5 A resolution

We report here the X-ray crystal structure of native subtilisin Carlsberg, solved at 2.5 A resolution by molecular replacement and refined by restrained least squares to a crystallographic residual (Formula see text): of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite 82 amino acid substitutions and one deletion in subtilisin Carlsberg relative to subtilisin BPN', the structures of these enzymes are remarkably similar. We calculate an r.m.s. difference between equivalent alpha-carbon positions in subtilisin Carlsberg and subtilisin BPN' of only 0.55 A. This confirms previous reports of extensive structural homology between these two subtilisins based on X-ray crystal structures of the complex of eglin-c with subtilisin Carlsberg [McPhalen, C.A., Schnebli, H.P. and James, M.N.G. (1985) FEBS Lett., 188, 55; Bode, W., Papamokos, E. and Musil, D. (1987) Eur. J. Biochem., 166, 673-692]. In addition, we find that the native active sites of subtilisins Carlsberg and BPN' are virtually identical. While conservative substitutions at residues 217 and 156 may have subtle effects on the environments of substrate-binding sites S1' and S1 respectively, we find no obvious structural correlate for reports that subtilisins Carlsberg and BPN' differ in their recognition of model substrates. In particular, we find no evidence that the hydrophobic binding pocket S1 in subtilisin Carlsberg is 'deeper', 'narrower' or 'less polar' than the corresponding binding site in subtilisin BPN'.     

Structural differences in the two major wheat germ agglutinin isolectins

We have combined amino acid sequence data with x-ray diffraction results to determine differences in structure of wheat germ agglutinin isolectin 1 (WGA1) relative to the known structure of wheat germ agglutinin isolectin 2 (WGA2). Electron density difference maps computed at 2.2 A resolution with coefficients [2F(WGA1) - F(WGA2)] and [F(WGA1) - F(WGA2)] and based on refined model phases of the WGA2 structure have revealed that the largest differences in the two isolectin structures are localized in the B-domain of the molecule. Amino acid sequence studies of tryptic and thermolytic peptides of WGA1 confirm the strong homology between the two isolectins and suggest variability at only four sequence positions. Three of these are closely spaced in domain B. The two histidines in WGA2, His59 and His66, are substituted by Gln and Tyr, respectively, and Pro56, by Thr in WGA1. The fourth difference at position 93 in domain C was identified as a change from Ser (WGA2) to Ala (WGA1). With these substitutions WGA1 exhibits a slightly higher degree of internal homology than does WGA2. In addition, we have carried out fluorescence studies on tryptic peptide T-3 to confirm the presence of a second Trp residue in the wheat germ agglutinin molecule, recently predicted at position 41 during the course of high resolution crystal structure refinement of WGA2.     

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the refined crystal structures of two wheat germ isolectins

The crystal structures of two closely related members of the multigene family of wheat lectins (isolectins 1 and 2) have been compared. These isolectins differ at five sequence positions, one being located in the saccharide binding site modulating ligand affinity. Crystals of the two isolectins are closely isomorphous (space group C2). The atomic models are based on structure refinement at 1.8 A resolution in the case of isolectin 2 (WGA2) and 2.0 A resolution in the case of isolectin 1 (WGA1). Refinement results for WGA1, recently completed with a crystallographic R-factor of 16.5% (Fo greater than 3 sigma (Fo)), are presented. Examination of a difference Fourier map, [FWGA2-FWGA1], at 2.0 A resolution and direct superposition of the two models indicated an overall close match of the two structures. Local differences are observed in the region of residues 44 to 69, where three sequence differences occur, and at highly mobile external residues on the surface. The average positional discrepancy (root-mean-square delta r) for corresponding protein atoms in the two crystal structures is 0.64 A for independent protomer I and 0.61 A for protomer II (0.29 A and 0.30 A for main-chain atoms). The mean atomic temperature factors are very similar 20.9 versus 22.0 A2). Regions of high flexibility coincide in the two isolectin structures. Of the 210 water sites identified in WGA1, 144 have corresponding positions in WGA2. A set of 51 well-ordered sites was found to be identical in the two independent environments in both structures, and was considered to be important for structure stabilization. Both of the unique sugar binding sites superimpose very closely, exhibiting root-mean-square positional differences ranging from 0.29 A to 0.42 A. The side-chains of the critical tyrosine residues, Tyr73 (P-site) and Tyr159 (S-site), superimpose best, while other highly flexible aromatic groups (Tyr64 and Trp150) and several water sites display large differences in position (0.5 to 1.0 A) and high temperature factors. The aromatic side-chains of Tyr66 in WGA1 and His66 in WGA2 are oriented similarly.     

A preliminary crystallographic study of wheat germ agglutinin

Wheat germ agglutinin crystallizes in two monoclinic space groups, P2₁ and C2, under identical crystallization conditions. Unit cell dimensions are $\text{a = 73.8}\text{A}\text{?}\text{, b = 51.2}\text{A}\text{?}\text{, c = 90.8}\text{A}\text{?}\text{, γ = 90 °}$ for $\text{P2}{}_1\text{; a = 51.31}\text{A}\text{?}\text{, b = 73.35}\text{A}\text{?}\text{, c = 91.45}\text{A}\text{?}\text{, β = 97.75 °}$ for C2, both with eight subunit molecules in the unit cell. The C2 crystals were chosen as suitable for investigating the three-dimensional structure to high resolution, because of their smaller asymmetric unit (containing the dimer), and also because they display better diffraction patterns.     

Fluorescence analysis of hormone binding activities of wheat germ agglutinin

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.