The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase.

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Accuracy of genomic prediction using an evenly spaced, low-density single nucleotide polymorphism panel in broiler chickens.

One approach for cost-effective implementation of genomic selection is to genotype training individuals with a high-density (HD) panel and selection candidates with an evenly spaced, low-density (ELD) panel. The purpose of this study was to evaluate the extent to which the ELD approach reduces the accuracy of genomic estimated breeding values (GEBV) in a broiler line, in which 1,091 breeders from 3 generations were used for training and 160 progeny of the third generation for validation. All birds were genotyped with an Illumina Infinium platform HD panel that included 20,541 segregating markers. Two subsets of HD markers, with 377 (ELD-1) or 766 (ELD-2) markers, were selected as ELD panels. The ELD-1 panel was genotyped using KBiosciences KASPar SNP genotyping chemistry, whereas the ELD-2 panel was simulated by adding markers from the HD panel to the ELD-1 panel. The training data set was used for 2 traits: BW at 35 d on both sexes and hen house production (HHP) between wk 28 and 54. Methods Bayes-A, -B, -C and genomic best linear unbiased prediction were used to estimate HD-marker effects. Two scenarios were used: (1) the 160 progeny were ELD-genotyped, and (2) the 160 progeny and their dams (117 birds) were ELD-genotyped. The missing HD genotypes in ELD-genotyped birds were imputed by a Gibbs sampler, capitalizing on linkage within families. In scenario (1), the correlation of GEBV for BW (HHP) of the 160 progeny based on observed HD versus imputed genotypes was greater than 0.94 (0.98) with the ELD-1 panel and greater than 0.97 (0.99) with the ELD-2 panel. In scenario (2), the correlation of GEBV for BW (HHP) was greater than 0.92 (0.96) with the ELD-1 panel and greater than 0.95 (0.98) with the ELD-2 panel. Hence, in a pedigreed population, genomic selection can be implemented by genotyping selection candidates with about 400 ELD markers with less than 6% loss in accuracy. This leads to substantial savings in genotyping costs, with little sacrifice in accuracy.

Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells.

The phytoestrogens genistein, daidzein and the daidzein metabolite equol have been shown previously to possess oestrogen agonist activity. However, following consumption of soya diets, they are found in the body not only as aglycones but also as metabolites conjugated at their 4'- and 7-hydroxyl groups with sulphate. This paper describes the effects of monosulphation on the oestrogen agonist properties of these three phytoestrogens in MCF-7 human breast cancer cells in terms of their relative ability to compete with [(3)H]oestradiol for binding to oestrogen receptor (ER), to induce a stably transfected oestrogen-responsive reporter gene (ERE-CAT) and to stimulate cell growth. In no case did sulphation abolish activity. The 4'-sulphation of genistein reduced oestrogen agonist activity to a small extent in whole-cell assays but increased the relative binding affinity to ER. The 7-sulphation of genistein, and also of equol, reduced oestrogen agonist activity substantially in all assays. By contrast, the position of monosulphation of daidzein acted in an opposing manner on oestrogen agonist activity. Sulphation at the 4'-position of daidzein resulted in a modest reduction in oestrogen agonist activity but sulphation of daidzein at the 7-position resulted in an increase in oestrogen agonist activity. Molecular modelling and docking studies suggested that the inverse effects of sulphation could be explained by the binding of daidzein into the ligand-binding domain of the ER in the opposite orientation compared with genistein and equol. This is the first report of sulphation enhancing activity of an isoflavone and inverse effects of sulphation between individual phytoestrogens.

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues.

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.

Twists and turns: a tale of two shikimate-pathway enzymes.

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

Phosphorylase recognition and phosphorolysis of its oligosaccharide substrate: answers to a long outstanding question.

Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-alpha-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 A resolution; and the 2.1 A resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites -1 to +4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites -1 and +1, the site of catalysis, from the preferred conformation for alpha(1-4)-linked glucosyl polymers.

Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication.

Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks.

The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex.

Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively. Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP. Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub-site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to target a number of different enzymes. The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells.

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.

The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.

A strategy for the incorporation of water molecules present in a ligand binding site into a three-dimensional quantitative structure--activity relationship analysis.

Water present in a ligand binding site of a protein has been recognized to play a major role in ligand-protein interactions. To date, rational drug design techniques do not usually incorporate the effect of these water molecules into the design strategy. This work represents a new strategy for including water molecules into a three-dimensional quantitative structure-activity relationship analysis using a set of glucose analogue inhibitors of glycogen phosphorylase (GP). In this series, the structures of the ligand-enzyme complexes have been solved by X-ray crystallography, and the positions of the ligands and the water molecules at the ligand binding site are known. For the structure-activity analysis, some water molecules adjacent to the ligands were included into an assembly which encompasses both the inhibitor and the water involved in the ligand-enzyme interaction. The mobility of some water molecules at the ligand binding site of GP gives rise to differences in the ligand-water assembly which have been accounted for using a simulation study involving force-field energy calculations. The assembly of ligand plus water was used in a GRID/GOLPE analysis, and the models obtained compare favorably with equivalent models when water was excluded. Both models were analyzed in detail and compared with the crystallographic structures of the ligand-enzyme complexes in order to evaluate their ability to reproduce the experimental observations. The results demonstrate that incorporation of water molecules into the analysis improves the predictive ability of the models and makes them easier to interpret. The information obtained from interpretation of the models is in good agreement with the conclusions derived from the structural analysis of the complexes and offers valuable insights into new characteristics of the ligands which may be exploited for the design of more potent inhibitors.

Oligosaccharide substrate binding in Escherichia coli maltodextrin phosphorylase.

The crystal structure of E. coli maltodextrin phosphorylase co-crystallized with an oligosaccharide has been solved at 3.0 A resolution, providing the first structure of an oligosaccharide bound at the catalytic site of an alpha-glucan phosphorylase. An induced fit mechanism brings together two domains across the catalytic site tunnel. A stacking interaction between the glucosyl residue and the aromatic group of a tyrosine residue at a sub-site remote (8 A) from the catalytic site provides a key element in substrate recognition; mutation of this residue to Ala decreases the Kcat/Km by 10(4). Extrapolation of the results to substrate binding across the site of attack by phosphorolysis indicates a likely alteration in the glycosidic torsion angles from their preferred values, an alteration that appears to be important for the catalytic mechanism.

The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.

In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.

The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor.

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the reciprocal activities of glycogen phosphorylase (GP) and glycogen synthase. An alkyl-dihydropyridine-dicarboxylic acid has been found to be a potent inhibitor of GP, and as such has potential to contribute to the regulation of glycogen metabolism in the non-insulin-dependent diabetes diseased state. The inhibitor has no structural similarity to the natural regulators of GP. We have carried out structural studies in order to elucidate the mechanism of inhibition. RESULTS: Kinetic studies with rabbit muscle glycogen phosphorylase b (GPb) show that the compound (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5, 6-tricarboxylate (Bay W1807) has a Ki = 1.6 nM and is a competitive inhibitor with respect to AMP. The structure of the cocrystallised GPb-W1807 complex has been determined at 100K to 2.3 A resolution and refined to an R factor of 0.198 (Rfree = 0.287). W1807 binds at the GPb allosteric effector site, the site which binds AMP, glucose-6-phosphate and a number of other phosphorylated ligands, and induces conformational changes that are characteristic of those observed with the naturally occurring allosteric inhibitor, glucose-6-phosphate. The dihydropyridine-5,6-dicarboxylate groups mimic the phosphate group of ligands that bind to the allosteric site and contact three arginine residues. CONCLUSIONS: The high affinity of W1807 for GP appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor results from the induced conformational changes that lock the enzyme in a conformation known as the T' state. Allosteric enzymes, such as GP, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compounds.

N-acetyl-beta-D-glucopyranosylamine: a potent T-state inhibitor of glycogen phosphorylase. A comparison with alpha-D-glucose.

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

Glucose analogue inhibitors of glycogen phosphorylase: from crystallographic analysis to drug prediction using GRID force-field and GOLPE variable selection.

Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.

Comparative molecular field analysis using GRID force-field and GOLPE variable selection methods in a study of inhibitors of glycogen phosphorylase b.

A primary goal in any drug design strategy is to predict the activity of new compounds. Comparative molecular field analysis (CoMFA) has been used in drug design and three-dimensional quantitative structure/activity relationship (3D-QSAR) methods. The CoMFA approach permits analysis of a large number of quantitative descriptors and uses chemometric methods such as partial least squares (PLS) to correlate changes in biological activity with changes in chemical structure. One of the characteristics of the 3D-QSAR method is the large number of variables which are generated in order to describe the nonbonded interaction energies between one or more probes and each drug molecule. Since it is difficult to know a priori which variables affect the biological activity of the compounds, much effort has been devoted to developing methods that optimize the selection of only those variables of importance. This work focuses on some of the aspects involved in the selection of such variables, applied to a series of glucose analogue inhibitors of glycogen phosphorylase b, using the program GRID to describe the molecular structures and using a method of generating optimal partial least squares estimations (program GOLPE) as the chemometric tool. This data set, consisting of over 30 compounds in which the three-dimensional ligand-enzyme bound structures are known, is well suited to study the effect of different data pretreatment procedures on the final model used for the prediction of new drug molecules. By relying on our knowledge of the real physical problem (i.e., using the combined crystallographic and kinetic results), it has been shown that suitable data pretreatment and variable selection have been found that does not result in a significant loss of relevant information. Moreover, by using an appropriate scaling procedure, GOLPE variable selection minimizes the risk of overfitting and overpredicting.

The design of potential antidiabetic drugs: experimental investigation of a number of beta-D-glucose analogue inhibitors of glycogen phosphorylase.

alpha-D-glucose is a weak inhibitor (Ki = 1.7 mM) of glycogen phosphorylase (GP) and acts as physiological regulator of hepatic glycogen metabolism; it binds to GP at the catalytic site and stabilizes the inactive T state of the enzyme promoting the action of protein phosphatase 1 and stimulating glycogen synthase. The three-dimensional structures of T state rabbit muscle GPb and the GPb-alpha-D-glucose complex have been exploited in the design of better regulators of GP that could shift the balance between glycogen synthesis and glycogen degradation in favour of the former. Close examination of the catalytic site with alpha-D-glucose bound shows that there is an empty pocket adjacent to the beta-1-C position. beta-D-glucose is a poorer inhibitor (Ki = 7.4 mM) than alpha-D-glucose, but mutarotation has prevented the binding of beta-D-glucose in T state GP crystals. A series of beta-D-glucose analogues has been designed and tested in kinetic and crystallographic experiments. Several compounds have been discovered that have an increased affinity for GP than the parent compound.