Molecular-Level Examination of Cu<sup>2+</sup> Binding Structure for Amyloid Fibrils of 40-Residue Alzheimer’s β by Solid-State NMR Spectroscopy

Rempel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2013

112

179

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ 42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ 42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ 42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu 2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.soluble Aβ oligomers | amyloid beta peptide | copper | electrospray ionization | Finke-Watsky mode P rotein aggregation is one of the immediate causes of Alzheimer's, Parkinson, and Huntington diseases, motivating biophysical studies of the responsible proteins. More than 20 small proteins undergo amyloidosis in humans. In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1, 2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 . Although the amyloid-cascade hypothesis suggests that the Aβ-containing amyloid plaques are responsible for neurodegeneration (3-7), other studies suggest that soluble aggregates of Aβ 42 are more neurotoxic than the amyloid plaques (8-13).The amyloid plaques in AD-affected brains contain high levels of copper, zinc, and iron (14-20). Among these, Cu has drawn the most attention because the Aβ precursor protein is likely a Cuchaperone protein (21). Several studies of Cu 2+ -Aβ 40 interactions show that Cu 2+ can promote Aβ 40 aggregation (14,18,19).The structure of Aβ 42 and its aggregates, although studied extensively, remains of high interest. Studies of amyloid fibrils invoke X-ray crystallography (22-24), EM (19,25,26), and thioflavin T fluorescence (19, 27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28-30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.MS should offer an opportunity for investigating soluble aggregates of Aβ 42 . Thus far, there are no MS-based, timedependent studies of the formation of soluble aggregates. Moreover, there are no ot...

Section: Resultsmentioning

confidence: 99%

“…Studies of amyloid fibrils invoke X-ray crystallography (22)(23)(24), EM (19,25,26), and thioflavin T fluorescence (19,27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28)(29)(30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.…”

mentioning

confidence: 99%

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Rempel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2013

112

179

Phys. Chem. Chem. Phys.

“…The assignment of the histidine aromatic carbon chemical shifts is in agreement with a recent study in which it was found that His-13, His-40 and the Val-40 carboxylic group are involved in binding to a Cu(II) metal ion. 49 Assuming that His-13 and His-14 are located in a b-sheet secondary structure element, His-14 is facing the solvent and cannot be involved in hydrogen bonds within one protofilament. The i, i À 2 and i, i + 2 neighboring residues Val-12 and Lys-16 are not potential hydrogen bonding partners.…”

Section: Resultsmentioning

confidence: 99%

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

Agarwal

Linser

Dasari

et al. 2013

The amyloid b-peptide (Ab) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Ab peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.Alzheimer's disease (AD) is a slowly progressive neurological disorder which is associated with memory loss, cognitive impairment and finally dementia and death. The amyloid b-peptide (Ab) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Ab is a cleavage product resulting from Alzheimer Precursor Protein (APP) processing. 1 The g-secretase cut yields Ab fragments of different lengths of which Ab40 and Ab42 are most abundant. 2,3 In vitro, the Ab peptide aggregates over time into oligomers and fibrillar structures. 4 In the past, several Ab fibril structure models have been suggested. These are based on MAS solidstate NMR experiments, 5-11 and cryo-electron microscopic image reconstructions. 12 Even though there is some controversy concerning the conformational space that Ab fibrils can adopt, all published models, including the 2-fold 7,10 and 3-fold 9 symmetric Ab 1-40 fibril structure suggested by Tycko, and Bertini and co-workers, agree on the basic building block which involves a b-sheet (b1, residues 12-24), a turn, and a second b-sheet (b2, residues 28-40). Biophysical studies indicate a certain degree of conformational plasticity for the N-terminal b-sheet. 13,14 Whereas b2 is present in all the studies, b1 can be truncated or does not adopt a regular b-sheet structure. Depending on the preparation conditions, amide protons in the region of the peptide where the first b-strand is expected show differential H/D protection factors. 13 Similarly, a certain degree of conformational variability in the N-terminus is suggested using cryo-electron microscopy. 14 In all structural models, Ab peptides are arranged in a parallel fashion. An antiparallel arrangement is only observed for the Iowa mutant Ab-D23N, 15 and for truncated forms of the peptide such as Ab [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . 16 The fibril structure is stabilized in particular by hydrogen bonding interactions. Therefore, observation of exchangeable and titratable groups is of particular interest to identify hydrogen bonding patterns. We and others could show in the past that high resolution proton spectra can be obtained for...

“…Furthermore, because the Cu 21 and His residues did not interact, the results from this MD simulation contradict those reported in the literature. 9,16,17 Consequently, we evaluated the amino acid residues that interacted with Cu 21 , Asp 23, and Glu 22, as displayed in Figure 4(B-D). The distance between Cu 21 and Asp 23 was lower (2.01 Å ) compared with that between Cu 21 and Glu22 (4.81 Å ) at the end of the simulation [ Fig.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

In silico and in vitro studies to elucidate the role of Cu²⁺ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

et al. 2013

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ1–42) is the most important pathophysiological event associated with Alzheimer's disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α‐helix to a β‐sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu2+ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu2+ and galanthamine prevent the formation of Aβ1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ1–42 acquires a characteristic U‐shape before the α‐helix to β‐sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ1−42 in the presence of Cu2+ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu2+) or to Lys 28 (galanthamine), which prevents Aβ1−42 from adopting the U‐characteristic conformation that allows the amino acids to transition to a β‐sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu2+ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.