Structural Insights into Aβ42 Oligomers Using Site-directed Spin Labeling

Abstract

Oligomerization of the 42-residue peptide Aβ42 plays a key role in the pathogenesis of Alzheimer disease. Despite great academic and medical interest, the structures of these oligomers have not been well characterized. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy is a powerful approach for studying structurally ill-defined systems, but its application in amyloid oligomer structure study has not been systematically explored. Here we report a comprehensive structural study on a toxic Aβ42 oligomer, called globulomer, using site-directed spin labeling complemented by other techniques. Transmission electron microscopy shows that these oligomers are globular structures with diameters of ∼7-8 nm. Circular dichroism shows primarily β-structures. X-ray powder diffraction suggests a highly ordered intrasheet hydrogen-bonding network and a heterogeneous intersheet packing. Residue-level mobility analysis on spin labels introduced at 14 different positions shows a structured state and a disordered state at all labeling sites. Side chain mobility analysis suggests that structural order increases from N- to C-terminal regions. Intermolecular distance measurements at 14 residue positions suggest that C-terminal residues Gly-29-Val-40 form a tightly packed core with intermolecular distances in a narrow range of 11.5-12.5 Å. These intermolecular distances rule out the existence of fibril-like parallel in-register β-structures and strongly suggest an antiparallel β-sheet arrangement in Aβ42 globulomers.

Antiparallel Triple-strand Architecture for Prefibrillar Aβ42 Oligomers

“…Distance measurements show two intermolecular distances at 9 -10 Å and 15-17 Å for every residue position, providing unambiguous support for the lack of parallel in-register ␤ structure in A␤42 oligomers. This work echoes a recent finding that A␤42 globulomers adopt structures distinct from fibrils (39).…”

“…Expression of GroES-ubiquitin-A␤42 in E. coli and their purification were performed as previously described in Ngo and Guo (38). Expression of Usp2cc and removal of fusion protein GU to prepare full-length A␤42 was performed as previously described in Gu et al (39). The purity of A␤42 fusion proteins was checked with SDS-PAGE.…”

Influence of genetic variability and external regulating factors on amyloid-beta peptide aggregation

“…Later, the research team of Eisenberg revealed the structure of an off-pathway, cylindrical oligomer of a segment of the amyloid-forming protein αB crystallin, that resembled a β-barrel composed of six antiparallel β-strands (72). We and other groups demonstrated that amyloidogenic proteins, such as Aβ, pass through an antiparallel β-sheet structured state, corresponding to oligomers, before undergoing a transition in structure to parallel β-sheet fibrils (58)(59)(60)(73)(74)(75). Recently, the group of Tycko demonstrated that Aβ , containing the FAD-linked Iowa D23N mutation and causing CAA, assembled into antiparallel β-sheet structured fibrils that were thermodynamically metastable and had a ribbon-like appearance (39).…”

“…Low CSF levels of Aβ , the Aβ peptide consisting of 42 amino acids, in combination with high levels of total tau and phosphorylated tau, are highly predictive biomarkers for AD (74). Other candidate biomarkers are now under study and are related to Aβ metabolism, neuronal and synaptic degeneration, inflammation, and oxidative stress (73). The relevance of an early diagnosis relies on the prospect that pharmacologic interventions will likely be more effective in generating clinical benefits if started early in AD progression, before neurodegeneration is already too advanced.…”

“…Another hot topic in the AD research field is the identification of biomarkers in the plasma and cerebrospinal fluid (CSF) that are capable of diagnosing AD or detecting brain alterations in an early disease stage, as clinical symptoms only appear on average 10-15 years after disease onset (73). Low CSF levels of Aβ , the Aβ peptide consisting of 42 amino acids, in combination with high levels of total tau and phosphorylated tau, are highly predictive biomarkers for AD (74).…”