Torleif Härd

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.

According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-beta (Abeta) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Abeta assemblies is accompanied by a conformational change toward a high content of beta-structure. Here, we report the solution structure of Abeta(1-40) in complex with the phage-display selected affibody protein Z(Abeta3), a binding protein of nanomolar affinity. Bound Abeta(1-40) features a beta-hairpin comprising residues 17-36, providing the first high-resolution structure of Abeta in beta conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Abeta. Z(Abeta3) stabilizes the beta-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Abeta hairpin within a large hydrophobic tunnel-like cavity. Consequently, Z(Abeta3) acts as a stoichiometric inhibitor of Abeta fibrillation. The selected Abeta conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.

Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.

A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.