Crystal structure of rabbit muscle glycogen phosphorylase a in complex with a potential hypoglycaemic drug at 2.0 A resolution

CP320626 has been identified as a potent inhibitor, synergistic with glucose, of human liver glycogen phosphorylase a (LGPa), a possible target for type 2 diabetes therapy. CP320626 is also a potent inhibitor of human muscle GPa. In order to elucidate the structural basis of the mechanism of CP320626 inhibition, the structures of T state rabbit muscle GPa (MGPa) in complex with glucose and in complex with both glucose and CP320626 were determined at 2.0 A resolution, and refined to crystallographic R values of 0.179 (R(free)=0.218) and 0.207 (R(free)=0.235), respectively. CP320626 binds at the new allosteric site, some 33 A from the catalytic site, where glucose binds. The binding of CP320626 to MGPa does not promote extensive conformational changes except for small shifts of the side chain atoms of residues R60, V64, and K191. Both CP320626 and glucose promote the less active T state, while structural comparisons of MGPa-glucose-CP320626 complex with LGPa complexed with a related compound (CP403700) and a glucose analogue inhibitor indicate that the residues of the new allosteric site, conserved in the two isozymes, show no significant differences in their positions.     

Structural basis of the synergistic inhibition of glycogen phosphorylase a by caffeine and a potential antidiabetic drug

Caffeine, an allosteric inhibitor of glycogen phosphorylase a (GPa), has been shown to act synergistically with the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate (W1807). The structure of GPa complexed with caffeine and W1807 has been determined at 100K to 2.3 A resolution, and refined to a crystallographic R value of 0.210 (Rfree = 0.257). The complex structure provides a rationale to understand the structural basis of the synergistic inhibition between W1807 and caffeine. W1807 binds tightly at the allosteric site, and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface which transform GPa to the T'-like state conformation already observed with GPa-glucose-W1807 complex. A disordering of the N-terminal tail occurs, while the loop of polypeptide chain containing residues 192-196 and residues 43'-49', from the symmetry related subunit, shift to accommodate W1807. Caffeine binds at the purine inhibitor site by intercalating between the two aromatic rings of Phe285 and Tyr613 and stabilises the location of the 280s loop in the T state conformation.     

The structure of glycogen phosphorylase alpha at 2.5 A resolution

The structure of the glucose-inhibited form of glycogen phosphorylase a has been determined at a resolution of 2.5 Å. With the aid of the primary sequence derived by Titani et al. (1977) for this enzyme, we have constructed an atomic model of the 97,400 molecular weight monomer. A substantial improvement in the electron density map over that reported previously (Fletterick et al., 1976b) was achieved by extension of the data set to 2.5 Å and the inclusion of three additional “heavy-atom” derivatives in the phasing procedure. Main-chain and side-chain electron density are clearly resolved in the map, allowing an unambiguous correlation with the published primary structure. The course of the polypeptide backbone in the C-terminal half of the molecule has been modified at two positions from that reported in the 3.0 Å resolution interpretation.The enzyme is clearly organized into two domains, both with $\text{α}\text{β}$ packing topology. The catalytic site lies in a crevice at the interface between the two domains. α-d-Glucose, which stabilizes the inactive (T) conformation in the parent crystal, is bound at this site in the C_(6′) chair equatorial conformation within 6 Å of the pyridoxal phosphate coenzyme which is covalently bound through the ?-amino group of lysine 679.The larger, N-terminal domain is differentiated by folding architecture and tertiary contacts into two lobes or subdomains which share the same β-sheet backbone: the predominantly helical glycogen storage (maltoheptaose binding) lobe and the N-terminal subdomain. The latter is involved in a variety of protein-protein interactions with the monomer related by the 2-fold axis of the physiological dimer, and contains the serine 14-phosphate moiety and the AMP (positive effector) binding site. The core of the second domain is the complex (βαβ)′ folding unit previously characterized as the nucleotide binding fold (Rao &; Rossmann, 1973).     

The crystal structure of phosphorylase beta at 6 A resolution

The determination of the crystal structure of phosphorylase b in the presence of IMP at 6 Å resolution is described. The structure determination is based on two heavy-atom isomorphous derivatives and their anomalous contributions. The molecular boundary is clearly distinguishable in the electron density map, except in the region of subunit-subunit contact about the crystallographic dyad axis, which is the symmetry axis of the dimer. The dimer molecule is roughly ellipsoidal in shape with dimensions 63 Å × 63 Å × 116 Å. There is a pronounced cavity on the enzyme surface but it is not yet known if this is a substrate binding site.     

The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution.

Elongation factor G (EF-G) catalyzes the translocation step of protein synthesis in bacteria, and like the other bacterial elongation factor, EF-Tu--whose structure is already known--it is a member of the GTPase superfamily. We have determined the crystal structure of EF-G--GDP from Thermus thermophilus. It is an elongated molecule whose large, N-terminal domain resembles the G domain of EF-Tu, except for a 90 residue insert, which covers a surface that is involved in nucleotide exchange in EF-Tu and other G proteins. The tertiary structures of the second domains of EF-G and EF-Tu are nearly identical, but the relative placement of the first two domains in EF-G--GDP resembles that seen in EF-Tu--GTP, not EF-Tu--GDP. The remaining three domains of EF-G look like RNA binding domains, and have no counterparts in EF-Tu.     

Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (alphabetagammadelta)(4) and a total MW of 1.3 x 10(6). PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 A resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 A resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (alphabetagammadelta)(4) PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.     

Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.     

FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 A resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site.     

Novel design principle validated: glucopyranosylidene-spiro-oxathiazole as new nanomolar inhibitor of glycogen phosphorylase, potential antidiabetic agent

2-Naphthyl-substituted glucopyranosylidene-spiro-oxathiazole prepared following a novel design principle was found to be the best known glucose analogue inhibitor of rabbit muscle glycogen phosphorylase b (K(i) 160 nM).     

The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.     

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.     

The 1.20 A resolution crystal structure of the aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition

The aminopeptidase from Aeromonas proteolytica (AAP) is a bridged bimetallic enzyme that removes the N-terminal amino acid from a peptide chain. To fully understand the metal roles in the reaction pathway of AAP we have solved the 1.20 A resolution crystal structure of native AAP (PDB ID = 1LOK). The high-quality electron density maps showed a single Tris molecule chelated to the active site Zn(2+), alternate side chain conformations for some side chains, a sodium ion that mediates a crystal contact, a surface thiocyanate ion, and several potential hydrogen atoms. In addition, the high precision of the atomic positions has led to insight into the protonation states of some of the active site amino acid side chains.     

New phosphate binding sites in the crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with phosphate and formycin A

Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the "standard" phosphate binding site.     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

The 2.0 A crystal structure of the ERalpha ligand-binding domain complexed with lasofoxifene

Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERalpha/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents.     

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 A resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non-crystallographic symmetry. The active site, nested at the interface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.     

Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine

The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E. coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution. The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme. The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug. The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain.     

Interaction of muscle glycogen phosphorylase B with methotrexate, folic and folinic acids

The interaction of rabbit skeletal muscle glycogen phosphorylase b with methotrexate, folic and folinic acids has been studied. Microscopic dissociation constant for the glycogen phosphorylase b--methotrexate complex determined by analytical ultracentrifugation is 0.43 mM. A subunit of glycogen phosphorylase b is shown to have two sites for methotrexate binding. AMP and FMN diminish the affinity of glycogen phosphorylase b to methotrexate, whereas glycogen does not influence the methotrexate binding to the enzyme. Methotrexate, folic and folinic acids are found to be inhibitors of the muscle glycogen phosphorylase b. The inhibition is reversible and characterized by positive kinetic cooperativity (the Hill coefficient exceeds one unity). The value of the pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: folic acid (0.65 mM), methotrexate (1.01 mM), folinic acid (3.7 mM). The antagonism between methotrexate, folic and folinic acids, on the one hand, and AMP and FMN, on the other, is revealed for their combined action.     

The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.