Templating mechanism of S100A9 amyloids on Aβ fibrillar surfaces during amyloid co-aggregation process was revealed by synergy of biophysical methods including charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides, and Aβ oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aβ peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods—mainly spectroscopy and imaging techniques—to characterize Aβ/Ni(II) interactions in vitro, for different Aβ variants: Aβ(1–40), Aβ(1–40)(H6A, H13A, H14A), Aβ(4–40), and Aβ(1–42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aβ monomers. Equimolar amounts of Ni(II) ions retard Aβ aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aβ binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aβ dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aβ monomers, while in a membrane-mimicking environment (SDS micelles) coil–coil helix interactions appear to be induced. For SDS-stabilized Aβ oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aβ aggregation processes that are involved in AD brain pathology.

Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from Aβ peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid selfassembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Å resolution when exposed to XFEL radiation, which is an improvement of 0.15 Å over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

Significance: pathogenic changes in γ-secretase activity, and its response to different drugs, can be greatly affected by changes in saturation of γ-secretase with its substrate. The molecular mechanism is unclear. Results: multiscale molecular dynamics studies show that saturation of γ-secretase with its substrate can result in parallel binding of different substrate molecules at the docking site and the active site. C-terminal domain of the second substrate can bind at cytosolic end of presenilin subunit while γ-secretase is still processing its first substrate. Such interactions can disrupt dynamic presenilin structures that regulate proteolytic steps. Similar disruptions in dynamic presenilin structures can be produced by different drugs and by different disease-causing mutations. Thus, the presented two-substrate mechanism, can explain toxic inhibition of γ-secretase activity and toxic increase in production of the longer, more hydrophobic, Aβ-proteins. Toxic aggregation between N-terminal domains of the two substrates is controlled by nicastrin ectodomain. Such aggregation is more likely to happen with C99-βCTF-APP than with C83-αCTF-APP substrate, which can explain, why β-secretase path is more pathogenic than α-secretase path. The binding of C99-βCTF-APP substrate to γ-secretase can be controlled by substrate-channeling between nicastrin ectodomain and β-secretase. Conclusions: The presented two-substrate mechanism can explain why different studies consistently show that increase in saturation of γ-secretase with its substrate can support pathogenic changes in different sporadic and familiar cases of the disease. Future drug-development strategies can target different physiological mechanisms that control the balance between cellular levels of γ-secretase activity and the total amyloid metabolism.