Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site.

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.

High-throughput production and structural characterization of libraries of self-assembly lipidic cubic phase materials.

A protocol is presented for the high-throughput (HT) production of lyotropic liquid crystalline phases from libraries of lipids and lipid mixtures using standard liquid dispensing robotics, implementing methods that circumvent the problems traditionally associated with handling the highly viscous cubic phase. In addition, the ability to structurally characterize lipidic phases and assess functionality for membrane proteins contained within cubic phases, in a HT manner, is demonstrated. The techniques are combined and exemplified using the application of membrane protein crystallization within lipidic cubic phases.

Crystal structure of the amyloid-ß p3 fragment provides a model for oligomer formation in Alzheimer's disease.

Alzheimer's disease is a progressive neurodegenerative disorder associated with the presence of amyloid-ß (Aß) peptide fibrillar plaques in the brain. However, current evidence suggests that soluble nonfibrillar Aß oligomers may be the major drivers of Aß-mediated synaptic dysfunction. Structural information on these Aß species has been very limited because of their noncrystalline and unstable nature. Here, we describe a crystal structure of amylogenic residues 18-41 of the Aß peptide (equivalent to the p3 α/γ-secretase fragment of amyloid precursor protein) presented within the CDR3 loop region of a shark Ig new antigen receptor (IgNAR) single variable domain antibody. The predominant oligomeric species is a tightly associated Aß dimer, with paired dimers forming a tetramer in the crystal caged within four IgNAR domains, preventing uncontrolled amyloid formation. Our structure correlates with independently observed features of small nonfibrillar Aß oligomers and reveals conserved elements consistent with residues and motifs predicted as critical in Aß folding and oligomerization, thus potentially providing a model system for nonfibrillar oligomer formation in Alzheimer's disease.

Alzheimer's amyloid-beta rescues yeast from hydroxide toxicity.

Amyloid-beta(Abeta42), which is known to be toxic to neuronal cells, protects yeast cells from severe sodium hydroxide toxicity. More than 85% cell death was caused by treatment with 1 mM NaOH and approximately 95% was observed at a 2 mM concentration. However, greater than 55% cells survived the treatment in the presence of Abeta42. A strong protective effect of the peptide was also evident from the differential staining of the treated culture with propidium iodide.

Substrate mediated reduction of copper-amyloid-beta complex in Alzheimer's disease.

X-Ray absorption near-edge spectroscopy (XANES) has been used to probe the substrate mediated reduction of Cu(2+) in Abeta-Cu(2+) complexes by ascorbate and the neurotoxin 6-hydroxydopamine (6-OHDA), however dopamine and, in particular, cholesterol are incapable of reducing soluble monomeric Abeta-Cu(2+) complexes.

The structure of the amyloid-beta peptide high-affinity copper II binding site in Alzheimer disease.

Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-beta (Abeta) protein bound primarily to copper ions. The evidence for an oxidative stress role of Abeta-Cu redox chemistry is still incomplete. Details of the copper binding site in Abeta may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of Abeta peptides complexed with Cu(2+) in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length Abeta-Cu(2+) peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the Abeta-Cu(2+) complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu(2+) binding site is consistent with the hypothesis that the redox activity of the metal ion bound to Abeta can lead to the formation of dityrosine-linked dimers found in AD.

Structural rationale for low-nanomolar binding of transition state mimics to a family GH3 beta-D-glucan glucohydrolase from barley.

The interactions of a transition state mimic anilinomethyl glucoimidazole (AmGlcIm), with a K(i) constant of 0.6 x 10(-)(9) M and a Gibbs free energy value of -53.5 kJ/mol, with a family GH3 beta-d-glucan glucohydrolase from barley have been analyzed crystallographically and by ab initio quantum mechanical modeling. AmGlcIm binds 3 times more tightly to the beta-d-glucan glucohydrolase than a previously investigated phenyl glucoimidazole. In the enzyme-AmGlcIm complex, an additional residue, Tyr253, and a water molecule positioned between subsites -1 and +1 are recruited for binding. Analyses of the two binary complexes reveal the following. (i) An intricate network exists in which hydrogen bonds between the enzyme's catalytic pocket residues Lys206, His207, Tyr253, Asp285, and Glu491 and the glucoimidazoles are shorter by 0.15-0.53 A, compared with distances of hydrogen bonds in the Michaelis complex. (ii) The "glucose" moiety of the glucoimidazoles adopts a (4)E conformation that is vital for the low-nanomolar binding. (iii) The N1 atoms of the glucoimidazoles are positioned nearly optimally for in-line protonation by the Oepsilon1 atom of the catalytic acid/base Glu491. (iv) The enzyme derives binding energies from both glycone and aglycone components of the glucoimidazoles. (iv) The prevalent libration motion of the two domains of the enzyme could play a significant role during induced fit closure in the active site. (v) Modeling based on the structural data predicts that protons could be positioned on the N1 atoms of the glucoimidazoles, and the catalytic acid/base Glu491 could carry an overall negative charge. (vi) The enzyme-AmGlcIm complex reveals the likely structure of an early transition state during hydrolysis. Finally, the high-resolution structures enabled us to define minimal structures of oligosaccharides attached to Asn221, Asn498, and Asn600 N-glycosylation sites.

Structure of the haemagglutinin-neuraminidase from human parainfluenza virus type III.

The three-dimensional structure of the haemagglutinin-neuraminidase (HN) from a human parainfluenza virus is described at ca 2.0 A resolution, both in native form and in complex with three substrate analogues. In support of earlier work on the structure of the homologous protein from the avian pathogen Newcastle disease virus (NDV), we observe a dimer of beta-propellers and find no evidence for spatially separated sites performing the receptor-binding and neuraminidase functions of the protein. As with the NDV HN, the active site of the HN of parainfluenza viruses is structurally flexible, suggesting that it may be able to switch between a receptor-binding state and a catalytic state. However, in contrast to the NDV structures, we observe no ligand-induced structural changes that extend beyond the active site and modify the dimer interface.

Three-dimensional structure of the barley beta-D-glucan glucohydrolase in complex with a transition state mimic.

Glucophenylimidazole (PheGlcIm), a tetrahydroimidazopyridine-type inhibitor and 4H3 conformer mimic of a glucoside, binds very tightly to a barley beta-d-glucan glucohydrolase, with a Ki constant of 2 x 10(-9) m and a DeltaG of 51 kJ mol(-1). PheGlcIm binds to the barley beta-d-glucan glucohydrolase approximately 2 x 10(5) times tighter than laminarin, which is the best non-synthetic ground-state substrate found so far for this enzyme, 10(6) times tighter than 4-nitrophenyl beta-d-glucopyranoside, and 2 x 10(7) tighter than glucose. The three-dimensional structure of the beta-d-glucan glucohydrolase with bound PheGlcIm indicates that the complex resembles a hypothetical transition state during the hydrolytic cycle, that the enzyme derives substrate binding energy from the "aglycone" portion of the ligand, and that it also reveals an anti-protonation trajectory for hydrolysis. Continuous electron densities at the 1.6 sigma level form between the three active site residues Asp95, His207, and Asp285, and the C6OH, C7OH, C8OH, and C9OH groups of PheGlcIm. These electron densities correspond to the most favorable interactions in the three-dimensional structure of the beta-d-glucan glucohydrolase-PheGlcIm complex and indicate atomic distances equal to or less than 2.55 A. The crystallographic data were corroborated with ab initio molecular orbital calculations. The data indicate that the 4E conformation of the glucose part of PheGlcIm is critical for tight binding and provide the first evidence for probable substrate distortion during catalysis by this enzyme.

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases.

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.

Structural studies of the resistance of influenza virus neuramindase to inhibitors.

Zanamivir and oseltamivir, specific inhibitors of influenza virus neuraminidase, have significantly different characteristics in resistance studies. In both cases resistance is known to arise through mutations in either the hemagglutinin or neuraminidase surface proteins. A new inhibitor under development by Biocryst Pharmaceuticals, BCX-1812, has both a guanidino group, as in zanamivir, and a bulky hydrophobic group, as in oseltamivir. Using influenza A/NWS/Tern/Australia/G70C/75 (H1N9), neuraminidase variants E119G and R292K have previously been selected by different inhibitors. The sensitivity of these variants to BCX-1812 has now been measured and found in both cases to be intermediate between those of zanamivir and oseltamivir. In addition, the X-ray crystal structures of the complexes of BCX-1812 with the wild type and the two mutant neuraminidases were determined. The ligand is bound in an identical manner in each structure, with a rearrangement of the side chain of E276 from its ligand-free position. A structural explanation of the mechanism of resistance of BCX-1812, relative to zanamivir and oseltamivir in particular, is provided.