A model of the transition state in the alkaline phosphatase reaction. (41/22435)

A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 A resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzyme-catalyzed reaction.  (+info)

Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity. (42/22435)

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.  (+info)

Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity. (43/22435)

Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined. The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus. Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75%. The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry. On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered. The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors. Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells.  (+info)

A statistical analysis of N- and O-glycan linkage conformations from crystallographic data. (44/22435)

We have generated a database of 639 glycosidic linkage structures by an exhaustive survey of the available crystallographic data for isolated oligosaccharides, glycoproteins, and glycan-binding proteins. For isolated oligosaccharides there is relatively little crystallographic data available. A much larger number of glycoprotein and glycan-binding protein structures have now been solved in which two or more linked monosaccharides can be resolved. In the majority of these cases, only a few residues can be seen. Using the 639 glycosidic linkage structures, we have identified one or more distinct conformers for all the linkages. The O5-C1-O-C(x)' torsion angles for all these distinct conformers appear to be determined chiefly by the exo-anomeric effect. The Manalpha1-6Man linkage appears to be less restrained than the others, showing a wide degree of dispersion outside the ranges of the defined conformers. The identification of distinct conformers for glyco-sidic linkages allows "average" glycan structures to be modeled and also allows the easy identification of distorted glycosidic linkages. Such an analysis shows that the interactions between IgG Fc and its own N-linked glycan result in severe distortion of the terminal Galbeta1-4GlcNAc linkage only, indicating the strong interactions that must be present between the Gal residue and the protein surface. The applicability of this crystallographic based analysis to glycan structures in solution is discussed. This database of linkagestructures should be a very useful reference tool in three-dimensional structure determinations.  (+info)

Automated MAD and MIR structure solution. (45/22435)

Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations.  (+info)

The structure of an entire noncovalent immunoglobulin kappa light-chain dimer (Bence-Jones protein) reveals a weak and unusual constant domains association. (46/22435)

Monoclonal free light chains secreted in immunoproliferative disorders are frequently involved in renal complications, including a specific proximal tubule impairment, Fanconi's syndrome. The latter is characterized in most cases by intracellular crystallization including a light-chain variable-domain fragment which resists lysosomal proteases. Bence-Jones protein (BJP) DEL was isolated from a patient with myeloma-associated Fanconi's syndrome. The crystal structure of this human kappa immunoglobulin light-chain noncovalent dimer was determined using molecular replacement with the structure of molecule REI, as the variable domain, and that of BJP LOC as the constant domain. To our knowledge, DEL is the first complete kappa BJP structure described to date. The R-factor is 20.7% at 2.8 A resolution. The BJP DEL dimer was compared with other light-chain dimers and with Fab fragments with a kappa light chain. Although the domain-folding pattern was similar, the relative positions of the constant domains differed. BJP DEL showed a noncanonical quaternary structural arrangement which may be attributable to the poor CL-CL affinity and lack of an interchain disulfide bridge, combined with the conformational editing effect of the crystal-packing forces. Our results suggest that, in the absence of a disulfide bridge, most BJP CLs are probably mobile in solution. This may explain their high susceptibility to proteases and the absence of naturally occurring crystals for these dimers. Furthermore, these findings of an unusual quaternary structure of an immunoglobulin light-chain association extend our knowledge about the large and highly diverse structures of the immunoglobulin superfamily.  (+info)

Ubiquinol:cytochrome c oxidoreductase. Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b. (47/22435)

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N', N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0. 06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.  (+info)

The flavin environment in old yellow enzyme. An evaluation of insights from spectroscopic and artificial flavin studies. (48/22435)

Spectroscopic and chemical modification studies of modified flavins bound to old yellow enzyme have led to predictions about the flavin environment of this enzyme. These studies analyzed solvent accessibility and hydrogen bonding patterns of particular flavin atoms, in addition to suggesting amino acid residues that are in close proximity to those atoms. Here, these studies are evaluated in the light of the crystal structure of old yellow enzyme to reveal that the spectroscopic and modified flavin results are generally consistent with the crystal structure. This highlights the fact that these are useful methods for studying flavin binding site structure. Although several of the inferred properties of the flavin environment are not consistent with the crystal structure, these discrepancies occurred in cases where an incorrect choice was made from among multiple plausible explanations for an experimental result. We conclude that modified flavin studies are powerful probes of flavin environment; however, it is risky to specify details of interactions, especially because of uncertainties due to induced charge delocalization in the flavin.  (+info)