Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

The formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimer’s disease (AD). It is therefore critical to characterize Aβ oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an Aβ oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an Aβ(1-42) tetramer. It comprises a β-sheet core made of six β-strands, connected by only two β-turns. The two faces of the β-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of Aβ(1-42), we prepared a sample enriched in Aβ(1-42) octamers, formed by two Aβ(1-42) tetramers facing each other forming a β-sandwich structure. Notably, samples enriched in Aβ(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β-sheets edges of the Aβ(1-42) tetramers and octamers.


Introduction
Substantial genetic evidence links the amyloid-β peptide (Aβ) to Alzheimer's disease (AD) (1). However, there is great controversy in establishing the exact Aβ form responsible for neurotoxicity. Aβ is obtained from a membrane protein, the amyloid precursor protein (APP), through the sequential cleavage of β -and γ -secretase (2). It is generally considered that, upon APP cleavage, Aβ is released to the extracellular environment. Due to its hydrophobic nature, Aβ then aggregates into multiple species, commonly referred to as soluble Aβ oligomers (3), which eventually evolve into Aβ fibrils (4)(5)(6)(7), the main component of amyloid plaques. However, by focusing exclusively on Aβ aggregation in solution, the membrane is overlooked. Indeed, a large number of studies have shown that the interaction of Aβ with the membrane results in the formation of membrane-associated oligomers, whose formation is considered to be directly responsible for compromising neuronal membrane integrity (8)(9)(10)(11)(12).
In 2016, we reported conditions to prepare homogenous and stable Aβ oligomers in membrane-mimicking environments (13). We found that their formation was specific for Aβ(1-42)-the Aβ variant most strongly linked to AD-, that they adopted a specific β -sheet structure, which is preserved in a lipid environment provided by bicelles, and that they incorporated into membranes exhibiting various types of porelike behaviour. Because of these properties, we named them β -barrel pore-forming Aβ(1-42) oligomers (βPFOs Aβ(1-42) ). Here we present the atomic structures of β PFOs  by NMR and MS and provide a mechanism for membrane disruption based on electrophysiology experiments and simulation studies in membranes.
Next, we used nuclear Overhauser effect spectroscopy (NOESY) to obtain longrange structural information. From the cross-peaks observed in the 3D NH-NH NOESY experiment, we identified 8 NOEs between β 1 and β 2 strands of the red Aβ(1-42) subunit and 7 NOEs between β 2 strand of the red Aβ(1-42) subunit and the β 3 strand of the green Aβ(1-42) subunit (Fig. 1C). The observation of intra-and 6 inter-subunit NOEs allowed us to establish the topology of an asymmetric dimer unit and to confirm that all the peaks detected in the 2D [ 1 H, 15 N]-TROSY spectrum belonged to the same oligomer. Moreover, we also detected three NOEs involving residues of the β 3 strand (Fig. 1C), which could be explained only if two asymmetric dimer units interacted through β 3 to form a tetramer in an antiparallel manner. All together, these NOEs allowed us to establish the complete topology of a six-stranded Aβ(1-42) tetramer unit (Fig. 1D) A for the backbone and the heavy atoms of the six-stranded β -sheet core, respectively. Notably, all residues on both faces of the β -sheet core were hydrophobic except for three basic residues (i.e., H13, H14, and K16) located in β 1, at the edges of the β -sheet core (Fig. 2B). On the 7 other hand, residues making the β -turns and the flexible N-termini ends were hydrophilic except for those comprising α 1.

Aβ(1-42) tetramer -DPC interaction
Having established the 3D structure and the physicochemical properties of the Aβ(1-42) tetramer, we examined how it interacted with the surrounding media, namely water and the DPC detergent molecules. 2D [ 1 H, 15 N]-HSQC spectra were acquired at two pH 8.5 and 9.5 ( fig. S8A,B). Residues belonging to the β -sheet core and some belonging to α 1 were detected at both pHs, while some of the α 1 residues and those corresponding to the β -turns and the N-termini ends were detected only when the spectrum was measured at the lowest pH. This observation thus suggests that residues comprising the β -turns and the N-termini ends exchanged faster with the solvent and were therefore more exposed than those making the β -sheet core and α 1 (Fig. 2C). To establish whether the more protected β -sheet core residues exhibited distinct degrees of solvent protection, we determined their amide temperature coefficients (Δδ/ΔT). Most of the NH amide protons of residues comprising β1, β2 and β3 were the most affected by temperature changes, which is consistent with these residues forming stable hydrogen bonds (14). In contrast, amide protons of β 1 residues pointing out of the β -sheet core (i.e., Y10, V12, H14, K16, V18, and F20) exhibited the lowest amide temperature coefficients, suggesting that these residues are the most water accessible of all residues comprising the β -sheet core (Fig. 2C).
Analysis of this spectrum allowed us to identify two types of intermolecular 8 interactions. First, we detected intermolecular NOEs between residues V12, L17, and L18, located in β 1, and the N-bound methyl groups of the choline head group of DPC ( Fig. 2D and fig. S9). Notably, this observation suggested that the detergent head group is bent towards the positively charged side chains (i.e., H13, H14, and K16) located at the hydrophilic edges of the β -sheet core in order to stabilize them.
Second, we detected intermolecular NOEs between all amide protons comprising the β -sheet core and the hydrophobic tail of DPC, with the largest intensities for residues located at the center of the β -sheet core and decreasing toward its edges ( Fig. 2D and   fig. S9). These observations were confirmed using a paramagnetic labeled detergent, 16-doxyl stearic acid (16-DSA) ( Fig. 2D and figs. S10, S11, and S12).
Finally, the interaction of the Aβ(1-42) tetramer with DPC micelles was further studied through molecular simulations using the SimShape approach (16). Over the course of a 1-ns simulation, the Aβ(1-42) tetramer was enveloped in a toroidal DPC micelle (fig. S13). Afterwards, the toroidal complex was equilibrated in explicit solvent for 60 ns. During this time, the hydrophobic terminal tail carbon of DPC was observed to interact predominantly with the two faces of the six-stranded β -sheet core, while transient contacts were also detected with the α 1 region. Additionally, the DPC polar head was observed to interact with the hydrophilic edges of the sixstranded β -sheet region, which slowly became exposed to the solvent (Fig. 2E).
Finally, these interactions were further validated by simulating the equilibrated protein-detergent complex in the absence of any external biasing forces ( fig. S14). In summary, the experimental and the simulation results indicate that both faces of the central hydrophobic β -sheet core of the Aβ(1-42) tetramer were covered with a monolayer of DPC with α 1 residues also interacting with the hydrophobic tail of DPC. In contrast, the rest of the residues, including the hydrophilic edges of the β -9 sheet core, were solvent-exposed and further stabilized by interactions with the polar head of DPC.

Aβ(1-42) tetramers and octamers are present in β PFOs Aβ(1-42) sample
Previous electrical recordings using planar lipid bilayers had revealed that the  . S16). Instead, although the detection of octameric forms required slightly higher activation conditions than tetramers, once detected, octamers did not break at the maximum activation conditions afforded by the instrument. This result indicated that octamers were not derived from the forced co-habitation of two tetramers in a micelle but rather from specific interactions between the Aβ subunits composing it.

Preparation of a β PFOs Aβ(1-42) sample enriched in Aβ(1-42) octamers
To pursue the characterization of octameric species, we attempted to enrich our sample in this oligomer form. To this end, we maintained the concentration of DPC micelles constant and increased the concentration of Aβ(1-42) to mimic the consequences of an increase in the latter in the membrane (12 3D). This analysis resulted in a major peak eluting 1.4 mL earlier than β PFOs LOW_Aβ(1-42), as well as a small peak eluting at the same volume as the major peak detected for β PFOs LOW_Aβ(1-42). These findings indicated that working at high Aβ(1-42) concentration indeed led to the formation of a larger oligomer.

1
To study the stoichiometry of the oligomers present in the two samples, after preparing them in DPC micelles without any buffer exchange, we submitted them to chemical crosslinking. Given the abundance of basic and acid moieties in the flexible regions of the Aβ(1-42) tetramer structure derived by NMR ( fig. S17), we decided to generate zero-length (ZL) cross-links between Lys and Asp or Glu residues using DMTMM as coupling reagent (16 To study the conformational state of the Aβ(1-42) octamers, we used IM-MS to derive their collision cross-sections ( TW CCS N2 ) (Fig. 4C-F). The experimental TW CCS N2 for the Aβ(1-42) tetramer was consistent only with the theoretical CCS obtained using the Aβ(1-42) tetramer structure determined by NMR, when the flexible loops were removed from the structure (Fig. 4C and 4E). This result 1 3 indicated that these residues were partially collapsed in the gas phase, in line with observations made for other membrane proteins containing flexible loops (18). The experimental TW CCS N2 for the Aβ(1-42) octamer was compared to two octamer models constructed using the 3D structure of the Aβ(1-42) tetramer as a building block (Fig. 4D). The first model was based on the association of two tetramers to form a loose β -barrel structure and the second one on the association of two
Therefore, our work widens the description of the much-needed low energy structural landscape of Aβ from Aβ fibrils. This landscape evolves from the 1 5 intermolecular formation of parallel β -sheets in Aβ fibrils, to intramolecular and intermolecular antiparallel β -sheet formation in the membrane-associated Aβ(1-42) oligomers reported in this work. By establishing the structure of membraneassociated Aβ(1-42) tetramers and octamers and assessing their activity in planar lipid bilayers and through MD simulations, we have revealed that their toxicity arises from the hydrophilic residues located on the edges of the β -sheets, which lead to the formation of lipid-stabilized pores. Such behavior resembles the toroidal pore-type behavior shown by many antimicrobial peptides (25) and would be consistent with the reported antimicrobial activity for Aβ (26,27). In summary, the present work represents the resolution of the first atomic structure of an Aβ membrane-associated oligomer and describes formation of lipid-stabilized pores as the potential mechanism underlying Aβ toxicity and its relation with AD. amino acid sequence of the Aβ(1-42) tetramer is arranged on the basis of the secondary and tertiary structure. Amino acids in square denote β-sheet secondary structure as identified by secondary chemical shifts; all other amino acids are in circles. Blue lines denote experimentally observed NOE contacts between two amide protons. Bold lines indicate strong NOEs typically observed between hydrogenbonded residues in β-sheets. Dashed lines show probable contacts between protons with degenerate 1 H chemical shifts. The side chains of white and gray residues point towards distinct sides of the β-sheet plane, respectively. Orange circles correspond to residues that could not be assigned.    The snapshots shown correspond to the initial coordinates (left), after 100 ns NPT equilibrium simulation (middle), and after 100 ns NVT simulation with 100 mV applied electric field (right). Protein is shown in grey, DPPC headgroup phosphorous atoms are shown in tan, and water in red/white.