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As molecular docking poses a challenging problem in rational drug design many workgroups have lavished their attention on finding and testing various docking techniques in order to maximize their benefits. One such experiment we report was conducted using the FlexX docking algorithm on a variety of data sets containing COX-2 inhibitors as well as randomly selected inactive molecules which were docked into the COX-2 crystal structure to test the predictive power of scoring functions available in either FlexX or CScore. Our goal was to determine both their abilities to separate active and inactive molecules, as well as to find which function shows the best correlation with the molecules' IC 50 values. We have shown, that F-score had the best ability to rank the COX-2 inhibitors according to their IC 50 values, while ChemScore has proven itself to be the most adequate for finding possible hits in large databases.

In several crystal structures of 3-phosphoglycerate kinase (PGK), the two domains occupy different relative positions. It is intriguing that the two extreme (open and closed) conforma- tions have never been observed for the enzyme from the same species. Furthermore, in certain cases, these different crystalline conformations represent the enzyme-ligand complex of the same composi- tion, such as the ternary complex containing either the substrate 3-phosphoglycerate (3-PG) and ,- imido-adenosine-5-triphosphate (AMP-PNP), an analogue of the substrate MgATP, or 3-PG and the product MgADP. Thus, the protein conformation in the crystal is apparently determined by the origin of the isolated enzyme: PGK from pig muscle has only been crystallized in open conformation, whereas PGK from either Thermotoga maritima or Trypano- soma brucei has only been reported in closed confor- mations. A systematic analysis of the underlying sequence differences at the crucial hinge regions of the molecule and in the protein-protein contact surfaces in the crystal, in two independent pairs of open and closed states, have revealed that 1) sequen- tial differences around the molecular hinges do not explain the appearance of fundamentally different conformations and 2) the species-specific intermo- lecular contacts between the nonconserved resi- dues are responsible for stabilizing one conforma- tion over the other in the crystalline state. A direct relationship between the steric position of the con- tacts in the three-dimensional structure and the conformational state of the protein has been demon- strated. Proteins 2004;55:198 -209.

Crystal structure of the ternary complex of pig muscle phosphoglycerate kinase (PGK) with the substrate 3-phosphoglycerate (3-PG) and the Mg(2+) complex of beta,gamma-methylene-adenosine-5'-triphosphate (AMP-PCP), a nonreactive analogue of the nucleotide substrate, MgATP, has been determined by X-ray diffraction at 2.5 A resolution. The overall structure of the protein exhibits an open conformation, similar to that of the previously determined ternary complex of the pig muscle enzyme with beta,gamma-imido-adenosine-5'-triphosphate (AMP-PNP) in place of AMP-PCP (May, Vas, Harlos, and Blake (1996) Proteins 24, 292-303). The orientation and details of interactions of the nucleotide phosphates, however, show marked differences. The beta-phosphate is linked to the conserved Asp 218, i.e., to the N-terminus of helix 8, through the Mg(2+) ion; the previously observed interactions of the metal complex of AMP-PNP or ADP with the conserved Asn 336 and the N-terminus of helix 13 are completely absent. These structural differences are maintained themselves in solution studies. Inhibition and binding experiments show a slightly weaker interaction of PGK with MgAMP-PCP than with MgAMP-PNP: at pH 7.5, the K(d) values are 1.07 +/- 0.18 and 0.41 +/- 0.08 mM, respectively. The difference is further enhanced by 3-PG: the K(d) values are 2.80 +/- 0.66 and 0.68 +/- 0.11 mM, respectively. Thus, the previously observed weakening effect of 3-PG on nucleotide binding (Merli, Szilágyi, Flachner, Rossi, and Vas (2002) Biochemistry 41, 111-119) is more pronounced with MgAMP-PCP. The discordance between substrate analogues also shows up in thiol reactivity studies. In their binary complexes, both ATP analogues protect the fast-reacting thiols of PGK in helix 13 against modification to similar extent. In their ternary complexes, however, which also contain bound 3-PG, the protective effect of MgAMP-PCP, but not of MgAMP-PNP, is largely abolished. This indicates a much smaller effect of MgAMP-PCP on the conformation of helix 13, which is in good correlation with its altered mode of phosphate binding and the ensuing increase in the flexibility of helix 13, as shown by elevated crystallographic B-factors. The possible existence of alternative site(s) for binding of the nucleotide phosphates may have functional relevance.

c ¨¨ ´ ´ ´ ´ ´ ´ Abstract Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies.We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins.Cellulase C has a TIM barrel fold with an additional helical subdomain.We designed and produced a number of mutants with the aim to increase its thermostability.Five mutants were designed to create new electrostatic interactions.They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme.Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains.In another mutant, the small helical subdomain was deleted.This mutant lost activity but its melting point was only 3 8C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function.A double mutant was designed to introduce a new disulfide bridge into the enzyme.This mutant is active and has an increased stability (DT s3 8C, m D(DG )s1.73 kcalymol) relative to the wild-type enzyme.Reduction of the disulfide bridge results in destabilization u and an altered thermal denaturation behavior.We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein.Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects. 2002 Elsevier Science B.V. All rights reserved.

A convenient resolution of racemic O,O′-dibenzoyltartaric acid by preferential crystallization of its mixed calcium salt formed with methoxyacetic acid is described. The separation of the partially prepared salt 2 followed by a subsequent recrystallization results in a simple and effective resolution of the title compound during which pure enantiomers of 1 can be obtained even on a large scale. The crystal structure of the conglomerate forming complex 2 is also reported.

Structures of chiral and achiral alcohol-O,O'-dibenzoyl-(2R,3R)-tartaric acid (DBTA) complexes were investigated by single-crystal X-ray diffraction (seven new crystal structures were determined). The complexes contain DBTA and chiral alcohol in 1:1, DBTA and achiral alcohol in 1:2 host-guest stoichiometry. The hydrogen bonding structures of chiral alcohol-DBTA and achiral alcohol-DBTA complexes are different, but within a subclass they are isostructural ones.

Three-dimensional model of the human histamine H1 receptor was developed by homology modelling using the high resolution structure of bovine rhodopsin as template. Genetic algorithm based docking calculations were used to identify the role of several amino acids having an effect on agonist or antagonist binding. Binding mode analyses of mepyramine, desloratidine, loratidine and acrivastine allowed us to rationalise their binding affinity. Binding site mapping resulted in seven new potential aromatic interaction points (Tyr 108, Phe 184, Phe 190, Phe 199, Phe 424, Trp 428, Tyr 431), that took part in forming the lipophilic pocket of the antagonist binding cavity.

A number of neurodegenerative diseases are mediated by mutation-induced protein misfolding. The resulting genetic defects, however, are expressed in varying phenotypes. Of the several well-established glycolytic enzyme deficiencies, triosephosphate isomerase (TPI) deficiency is the only one in which haemolytic anaemia is coupled with progressive, severe neurological disorder. In a Hungarian family with severe decrease in TPI activity, two germ line-identical but phenotypically differing compound heterozygote brothers inherited two independent (Phe240 → Leu and Glu145 → stop codon) mutations. We have demonstrated recently [Orosz, Oláh, Alvarez, Keserü, Szabó, Wágner, Kovári, Horányi, Baróti, Martial, Hollán and Ovádi (2001) Blood 98, 3106–3112] that the mutations of TPI explain in themselves neither the severe decrease in the enzyme activity characteristic of TPI deficiency nor the enhanced ability of the mutant enzyme from haemolysate of the propositus to associate with subcellular particles. Here we present kinetic (flux analysis), thermodynamic (microcalorimetry and fluorescence spectroscopy), structural (in silico) and ultrastructural (immunoelectron microscopy) data for characterization of mutant isomerase structures and for the TPI-related metabolic processes in normal and deficient cells. The relationships between mutation-induced TPI misfolding and formation of aberrant protein aggregates are discussed.

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line–identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates. Enhanced attachment of mutant TPI to erythrocyte inside-out vesicles and to microtubules of brain cells was found when the binding was measured with TPIs in hemolysate. In contrast, there was no difference between the binding of the recombinant wild-type and Phe240Leu mutant enzymes. These findings suggest that the missense mutation by itself is not enough to explain the low catalytic activity and “stickiness” of mutant TPI observed in hemolysate. The activity of the mutant TPI is further reduced by its attachment to inside-out vesicles or microtubules. Comparative studies of the hemolysate from a British patient with Glu104Asp homozygosity and with the platelet lysates from the Hungarian family suggest that the microcompartmentation of TPI is not unique for the hemolysates from the Hungarian TPI-deficient brothers. The possible role of cellular components, other than the mutant enzymes, in the distinct behavior of TPI in isolated form versus in hemolysates from the compound heterozygotes and the simple heterozygote family members is discussed.

The complexes of pig muscle 3-phosphoglycerate kinase with the substrate MgATP and with the nonsubstrate Mg(2+)-free ATP have been characterized by binding, kinetic, and crystallographic studies. Comparative experiments with ADP and MgADP have also been carried out. In contrast to the less specific and largely ionic binding of Mg(2+)-free ATP and ADP, specific occupation of the adenosine binding pocket by MgATP and MgADP has been revealed by displacement experiments with adenosine and anions, as well as supported by isothermal calorimetric titrations. The Mg(2+)-free nucleotides similarly stabilize the overall protein structure and restrict the conformational flexibility around the reactive thiol groups of helix 13, as observed by differential scanning microcalorimetry and thiol reactivity studies, respectively. The metal complexes, however, behave differently. MgADP, but not MgATP, further increases the conformational stability with respect to its Mg(2+)-free form, which indicates their different modes of binding to the enzyme. Crystal structures of the binary complexes of the enzyme with MgATP and with ATP (2.1 and 1.9 A resolution, respectively) have shown that the orientation and interaction of phosphates of MgATP largely differ not only from those of ATP but also from the previously determined ones of either MgADP [Davies, G. J., Gamblin, S. J., Littlechild, J. A., Dauter, Z., Wilson, K. S., and Watson, H. C. (1994) Acta Crystallogr. D50, 202-209] or the metal complexes of AMP-PNP [May, A., Vas, M., Harlos, K., and Blake, C. C. F. (1996) Proteins 24, 292-303; Auerbach, G., Huber, R., Grattinger, M., Zaiss, K., Schurig, H., Jaenicke, R., and Jacob, U. (1997) Structure 5, 1475-1483] and are more similar to the interactions formed with MgAMP-PCP [Kovári, Z., Flachner, B., Náray-Szabó, G., and Vas, M. (2002) Biochemistry 41, 8796-8806]. Mg(2+) is liganded to both beta- and gamma-phosphates of ATP, while beta-phosphate is linked to the conserved Asp218, i.e., to the N-terminus of helix 8, through a water molecule; the known interactions of either MgADP or the metal complexes of AMP-PNP with the N-terminus of helix 13 and with Asn336 of beta-strand J are absent in the case of MgATP. Fluctuation of MgATP phosphates between two alternative sites has been proposed to facilitate the correct positioning of the mobile side chain of Lys215, and the catalytically competent active site is thereby completed.

The energetics of the stereoselective reduction of norbornan-7-one derivatives have been studied by semiempirical AM1 molecular orbital calculations. It was found that the reaction is kinetically controlled; correct prediction of the selectivity is possible only on the basis of the relative energies of the transition states of the reaction. Theoretically calculated and experimental anti–syn product ratios are in semiquantitative agreement, in contrast to results obtained from molecular electrostatic potentials. Geometry relaxation of the transition state is essential in obtaining reliable isomer ratios.

Two-step alkylations of benzimidazole, 2-methylbenzimidazole, and imidazole at their nitrogen atoms with various bifunctional alkylating agents have afforded a series of 16-membered quaternary azacyclophanes. Of these macrocycles, those bearing methyl or methoxy groups on the azole or at the aromatic bridges have been found to exist as mixtures of conformers. In some cases, the structures of the conformers have been determined by NMR methods and X-ray crystallography, and have been correlated with the results of a computer-aided conformational search.