Insights into Formation and Structure of Aβ Oligomers Cross-Linked

May 8, 2017 - Department of Physics, Drexel University, Philadelphia, Pennsylvania 19104, United States. ‡ Faculty of Mathematics and Physics, Unive...
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Insights into Formation and Structure of A# Oligomers Cross-Linked via Tyrosines Shuting Zhang, Dillion M. Fox, and Brigita Urbanc J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

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Insights Into Formation And Structure of Aβ Oligomers Cross-Linked Via Tyrosines Shuting Zhang,∗,† Dillion M. Fox,†,¶ and Brigita Urbanc∗,†,‡ †Department of Physics, Drexel University, Philadelphia, PA 19104, USA ‡Faculty of Mathematics and Physics, University of Ljubljana, Slovenia ¶Current address: Department of Physics, University of Pennsylvania, Philadelphia, PA 19104, USA E-mail: [email protected]; [email protected] Fax: (215) 895-5934

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Abstract Alzheimer’s disease (AD) pathology is hypothesized to be triggered by amyloid βprotein (Aβ) assembly into oligomers. Oligomer size distributions of both predominant Aβ alloforms, Aβ40 and Aβ42 , can be determined in vitro using cross-linking followed by gel electrophoresis. Cross-linking, which can occur in vivo in the presence of copper and hydrogen peroxide, was recently shown to stabilize Aβ oligomers by inhibiting their conversion into fibrils. Whereas several studies showed that cross-linking is facilitated by dityrosine bond formation, the molecular-level mechanism of cross-linking remains unclear. Here, we use efficient discrete molecular dynamics with DMD4B-HYDRA force field to examine the effect of cross-linking via tyrosines on Aβ oligomer formation. Our results show that cross-linking via tyrosines promotes Aβ self-assembly, in particular that of Aβ40 , but does not account for cross-linked oligomers larger than Aβ40 trimers and Aβ42 tetramers. Cross-linking via tyrosines profoundly alters Aβ40 and Aβ42 oligomer conformations by increasing the solvent exposure of hydrophobic residues, resulting in elongated oligomeric morphologies that differ from globular structures of noncross-linked oligomers. When compared to available experimental data, our findings imply that amino acids other than tyrosines are involved in Aβ cross-linking, a proposition that is currently under investigation.

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Introduction Alzheimer’s disease (AD) is a neurodegenerative disease characterized by irreversible, progressive deterioration of learning and memory. 1,2 The hallmarks of AD are extracellular amyloid plaques, intracellular neurofibrillary tangles, and massive neuronal loss. The main component of the amyloid plaques is amyloid β-protein (Aβ), which is formed by sequential β-secretase and γ-secretase cleavages of amyloid precursor protein (APP). 3 The original amyloid cascade hypothesis implicated Aβ aggregation into insoluble fibrils and their deposition into amyloid plaques as the cause of AD. 4 Since then compelling evidence has accumulated demonstrating that soluble, low molecular weight (LMW) Aβ assemblies are the proximate neurotoxic species in AD. 5,6 This caused a paradigm shift from insoluble Aβ fibrils with a well-defined cross-β structure to relatively unstructured Aβ oligomers. 7 It is puzzling that of the two predominant species of Aβ, Aβ40 and Aβ42 , which structurally differ only by the presence of a short hydrophobic dipeptide, I41 A42 , at the C-terminus of Aβ42 , Aβ42 is more strongly associated with AD pathology than Aβ40 and forms assemblies that are more toxic to cell cultures and in vivo than assemblies formed by Aβ40 . 8–10 Clearly, characterization of structural differences between Aβ40 and Aβ42 oligomers is critical for understanding their distinct toxic function. Aβ oligomers can be best defined as metastable, non-covalently bonded assemblies, in general without any ordered structure. Their heterogeneous nature, lack of structural order, and relatively short lifetimes makes the application of classical, high-resolution methods for their structural characterization, such as X-ray crystallography and NMR, very challenging. Although Aβ18−41 tetramer was crystallized in a complex with 4 shark Ig new antigen receptor proteins, 11 to date no in vitro study has reported a 3D structure of oligomers formed by full-length Aβ40 or Aβ42 . Effective experimental techniques to determine the oligomer size distribution of Aβ revolve around cross-linking techniques, such as photo-induced crosslinking of unmodified proteins (PICUP) 12,13 and copper and hydrogen peroxide induced cross-linking of unmodified proteins (CHICUP), 14 followed by a separation method, such as 3

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro study by Bitan et al. was the first to introduce PICUP, followed by SDSPAGE to demonstrate that Aβ40 and Aβ42 oligomerize through distinct pathways, 12,15 a result supported also by studies using ion mobility combined with mass spectroscopy, which are independent of the PICUP chemistry. 16,17 PICUP utilizes ruthenium complex, ammonium persulfate, and light to facilitate cross-linking, conditions that are not present in vivo. Although PICUP cross-linked oligomers of Aβ40 have been isolated and structurally characterized in vitro, 18 it is not clear to which extent cross-linking alters Aβ oligomer structure. The recently reported alternative to PICUP, namely CHICUP, that does not require any light to initiate cross-linking, occurs in the presence of copper and hydrogen peroxide, which are both native to AD brain. 14 Both PICUP and CHICUP were shown to stabilize Aβ40 and Aβ42 oligomers, which consequently exert prolonged damage to biomimetic lipid vesicles. 14 CHICUP cross-linked Aβ oligomers may thus represent the proximate neurotoxic species in AD. 19 If so, examining the effect of cross-linking on oligomer formation and structure may provide important insights relevant to AD. Molecular dynamics (MD) offers a way to visualize and quantify protein structures at atomistic resolution, which is particularly important in studies of oligomers formed by intrinsically disordered proteins, such as Aβ, 20 α-synuclein, amylin, tau, and others. Fully atomistic MD, which utilizes explicit models of Aβ and solvent, is computationally untenable because of the large size of the system and long timescales required to study Aβ assembly. Simulations can be sped up using efficient discrete molecular dynamics (DMD) combined with a coarse-grain protein model in implicit solvent, which significantly reduces the number of atoms in each simulation and makes assembly timescales computationally accessible. The DMD4B-HYDRA approach 21 that utilizes DMD in combination with a fourbead protein model 22 and amino acid-specific interactions 23 has been successfully applied to study oligomer formation of Aβ40 and Aβ42 21,24 as well as their naturally occurring isoforms, 25–28 folding of mucin domains, 29 and oligomer formation by a globular amyloidogenic

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protein stefin B. 30 The DMD4B-HYDRA approach captures the distinct experimentallyderived oligomer size distributions 31 of several Aβ variants and offers several structural predictions 21,26–28,32 that are consistent with available experimental constraints and lead to structure-toxicity hypothesis linking flexible and solvent exposed N-termini of Aβ oligomers to their enhanced toxicity. 19 Here, we use the same DMD4B-HYDRA approach to examine the effect of cross-linking on Aβ40 and Aβ42 oligomer formation and structure and discuss implications of our findings for understanding the mechanism of cross-linking and its relevance to AD pathology.

Methods DMD4B-HYDRA Simulations Discrete Molecular Dynamics The continuous interparticle potentials used in molecular dynamics (MD) can be approximated by one or multiple square wells, which reduces the dynamics process into event-driven discrete molecular dynamics (DMD). 33–35 DMD is driven by collisions between pairs of particles. Between two successive collisions, particles perform constant-velocity motion, which eliminates the need to perform numerical integration. The DMD algorithm involves sorting of collision times in order to select the next collision event, which can be optimized using a priority queue. 36 Consequently, DMD can be up to several orders of magnitude faster than MD. Four-Bead Protein Model with Backbone Hydrogen Bonding In the four-bead model, 22 each amino acid (except glycine) is represented by four beads, which correspond to the amide (N), α carbon (Cα ) and the carbonyl (C) groups forming the peptide backbone as well as one side-chain bead corresponding to β carbon (Cβ ). This model uses the smallest number of beads that preserve the chiral nature of an individual amino acid. The length of bonds and the angles between pairs of bonds were calculated from the known folded protein

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structures of ∼7,700 proteins from the Protein Data Bank (PDB). 37 The effective hydrogen bond is formed between the amide bead Ni of amino acid i and the carbonyl bead Cj of amino acid j (i 6= j) with bond length in the range [4.0 ˚ A, 4.2 ˚ A]. The interaction strength of hydrogen bond is EHB , which represents a unit of energy in our simulation. The unit of temperature is EHB /kB . Our simulations are conducted at T = 0.13, which was previously shown to be a good estimate of physiological temperature, 26 which results in an estimate of the hydrogen bond energy of EHB = 4.7 kcal/mol. Amino Acid-Specific Interactions Due to Hydropathy and Charge In the DMD4BHYDRA force field, 21,23 the interactions among the side-chain beads are described using the phenomenological hydropathy scale by Kyte and Doolittle. 38 According to this hydropathy scale, at neutral pH, amino acids I, V, F, L, M, C, and A are hydrophobic; N, Q, and H are hydrophilic; R, D, K, and E are charged hydrophilic. Apart from G, which lacks the side-chain bead in this model, the remaining amino acids (T, S, W, Y, P) are treated as neutral and subjected to excluded volume interactions only. The Cβ -Cβ interactions are only allowed within the group of hydrophilic residues and within the group of hydrophobic residues. The remaining Cβ -Cβ interactions are due to excluded volume interaction. The effective hydropathic interaction is modeled by a single square-well potential with an interaction range of 0.75 nm. The absolute value of the effective hydropathic interaction strength is EHP = 0.3. The electrostatic interaction between two charged side-chain beads is implemented in the DMD4B-HYDRA force field through a double repulsive or attractive square well potential with an inner and outer interaction ranges of 0.6 nm and 0.75 nm, respectively. 23 In our simulations, the effective electrostatic interaction strength is ECH = 0. This choice is based on the results of a previous study, 26 which demonstrated that non-zero values of ECH cause a shift in the Aβ40 and Aβ42 oligomer size distributions to larger oligomer orders in a way that is inconsistent with the experimentally-derived (PICUP/SDS-PAGE) distributions. 12

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Implementation of Cross-Linking When the system is subjected to cross-linking, tyrosines at position 10 can form covalent bonds with each other. A covalent bond between two tyrosines is modelled as a square-well potential between two Cβ beads of tyrosines (Y) with an interaction strength of EY Y = 20, which corresponds to an estimate of a covalent bond strength of EY Y = 95 kcal/mol, and interaction range of 4.2 ˚ A. Because there is a single Y in each Aβ40 or Aβ42, each cross-link (covalent bond) is intermolecular. Each tyrosine can form multiple non-directional covalent bonds with other tyrosines. Whereas the non-directional nature of YY cross-linking is an approximation, our implementation of cross-linking is consistent with an observation that a single tyrosine can form two covalent bonds 39 and a more recent detection of bioactive tri- and even tetra-tyrosines. 40 Simulation Protocol Initial states for the 8 trajectories were generated by enclosing 32 peptides (Aβ40 or Aβ42 ) in a cubic simulation box of 25 nm, followed by a short hightemperature DMD trajectory, in which the force field was turned off to prevent any interactions, generating 8 independent states, each containing 32 spatially separated unstructured peptides. The 8 production runs (trajectories) were conducted at constant volume and temperature of T = 0.13, 26 which was an estimate of the physiological temperature T = 310K using the Berendsen thermostat. 41 From this temperature and the definition of q temperature, 12 mvx2 = 12 kB T , the average velocity of beads can be calculated: vx = kBmT . Here, m corresponds to the unit mass in DMD simulations. Each of the beads in the four-bead model had the same unit mass. For example, the mass of alanine is 4m. We could thus estimate m = 1/4MA = 3 × 10−26 kg, which resulted in vx ≈ 300m/s and ∆t = ∆x/vx = 10−11 /300 ≈ 0.03ps. DMD simulations were performed in two stages. In the first stage, each of the 8 trajectories was subjected to 40 × 106 time units (approximately 1.2 µs) long simulations, in which no cross-linking was allowed. In the second stage, two types of simulations of 50 × 106 time units (approximately 1.5 µs) length were conducted, both of them starting from the resulting confirmation of the first stage. One type of the simulations

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allowed the formation of cross-linking in addition to the conditions in the first stage. The other type of simulations were performed under the same conditions as in the first stage, as a control set.

Structural Analysis All the structural analysis was performed by coding within Virtual Molecular Dynamics (VMD) software package. 42 VMD was also used to visualize oligomer conformations. Time Evolution of Cross-linking. In order to investigate the time evolution of crosslinking, we calculated the number of covalent bonds among tyrosines as well as the number of non-bonded (“free”) tyrosines as a function of simulation time, using time frames that were 105 time units apart. At each time frame, the number of YY bonds and the number of free tyrosines were calculated for each of the eight ensembles, then the averages and the standard error of the mean (SEM) values were derived from the statistics over the 8 trajectories. Oligomer Size Distribution. When calculating the oligomer size distribution, which did not differentiate between non-covalently bonded and covalently bonded oligomers, we defined an oligomer by a proximity between any two peptides, regardless of whether the two peptides were cross-linked or not. If any two beads belonging to two different peptides were at a distance ≤ 5 ˚ A, the two peptides were considered to belong to the same oligomer. The oligomer size distributions and the corresponding SEM values were calculated every 10 × 106 time units. To get the average oligomer size distribution at simulation time T , individual oligomer size distributions (calculated for each time frame of each trajectory) were first averaged within each trajectory over three time frames (at T − 106 , T − 0.5 × 106 , and T ) and then the average and SEM values were computed using the eight resulting statisticallyindependent oligomer size distributions.

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Characterization of Sizes of Covalently Bonded Oligomers. In addition to the oligomer size distribution, which did not distinguish between covalently and non-covalently bonded oligomers, we also calculated distribution of oligomers that were bonded exclusively through YY cross-links (covalent bonds). Because there is only one tyrosine in each peptide, only intermolecular cross-links could form. Thus, every covalent bond between tyrosines contributed to formation of covalently-bonded (cross-linked) oligomers. Time frames between 30 × 106 and 50 × 106 time units within each cross-linking trajectory that were 105 time units apart (200 frames per trajectory) were used to extract the cross-linked oligomers. The total number of cross-linked oligomers of each order (size) was then calculated for Aβ40 and Aβ42 ensembles. Secondary Structure. The secondary structure characterization that accounted for αhelical, β-strand, turn, coil, and bridge propensities was derived using the STRIDE algorithm 43 implemented in VMD. 42 In our analysis we examined more closely the propensities for the three predominant secondary structures: β-strand, turn, and coil. We calculated βstrand, turn, and coil propensities per amino acid in Aβ40 and Aβ42 monomers and oligomers both for control and cross-linking ensembles. We also computed the average secondary structure content and the corresponding SEM values by, averaging the corresponding propensities over all peptides in the oligomer, and then over all oligomer conformations derived from time frames between 30×106 and 50×106 time units of each trajectory, again for control ensembles and cross-linking ensembles. Contact Maps. Two amino acids were considered to be in contact when the distance between their Cα or Cβ atoms was ≤ 0.5 nm. The contact map was calculated as a matrix of normalized contact frequencies between all pairs of residues in the sequence as well as the corresponding SEM values. The average contact frequencies are displayed in the upper left triangle of the contact map and the corresponding SEM values are shown in lower right triangle of the contact map. We distinguished tertiary and quaternary contacts when 9

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calculating the contact maps. When two amino acids belong to the same peptide, the contact between them contributed to the tertiary contact map. Otherwise, the contact contributed to quaternary contact map. The number of tertiary and quaternary contacts between Cα or Cβ atoms was calculated for each assembly state by averaging the number of contacts over all peptides in the assembly, followed by averaging over all conformations under study. Coarse-Grained (CG) Solvent Accessible Surface Area (SASA). We calculated the amino acid-specific CG-SASA values for each amino acid using the SASA calculation implemented in VMD. When calculating CG-SASA of an atom, a spherical surface at a distance of 1.4 ˚ Afrom its van der Waals surface was considered and the surface area that did not overlap with the surface area of any other atoms contributed to the CG-SASA value calculation. In addition to the four beads of each amino acid in the four-bead model, the backbone carbonyl oxygen and amide hydrogen were also included in calculation. The final CG-SASA of an amino acid was calculated by summing over “free” surface areas of all atoms within the amino acid. The average CG-SASA values per amino acid and the corresponding SEM values were calculated by averaging over all peptides and over all ensembles or conformations under study. Potential of Mean Force (PMF). The potential of mean force was calculated based on the distribution of conformations with respect to two reaction coordinates: X1 , the distance between N-terminal Cα atoms and the center of mass of the peptide (N-CM distance) and X2 , the sum of coarse-grained solvent-accessible surface area (CG-SASA) values over all hydrophobic residues (hydrophobic CG-SASA). The PMF values were calculated as βW (X1, X2 ) = ln P (X1 , X2 ), where P (X1, X2 ) is the normalized distribution of conformations projected onto the two reaction coordinates. First, for each peptide in the conformation, N-CM distance and hydrophobic CG-SASA were calculated and normalized over all peptides in the conformation. From the reaction coordinate values for each conformation in the ensemble under consideration, a two-dimensional histogram (with bin size of 10

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0.1 nm × 0.3 nm2 ) was constructed and normalized by the total number of peptides under study to get P (X). Finally, the PMF values were computed. We also calculated the corresponding one-dimensional distributions of N-CM distances and hydrophobic CG-SASA values. Mann-Whitney U-test 44 was performed to test whether cross-linking altered either of the two one-dimensional distributions. The lowest PMF values represented the most likely conformations with the lowest free energy. Representative conformations that corresponded to PMF minima were randomly selected for visualization.

Results and Discussion The purpose of this computational study is to examine the effect of cross-linking on oligomer formation and structure of the two predominant Aβ alloforms, Aβ40 and Aβ42 . The primary structure of Aβ1−42 is: 1

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The shorter alloform, Aβ1−40 , lacks the two C-terminal amino acids I41 A42 . The focus of our study is covalent cross-linking of soluble Aβ40 and Aβ42 as induced by PICUP 12,13,15 or CHICUP 14 as the two cross-linking techniques were shown to produce similar Aβ40 and Aβ42 oligomer size distributions. 14 Covalent cross-linking of protein complexes via PICUP was introduced and examined by Fancy and Kodadek, who reported that tyrosines and tryptophans are involved in formation of covalent bonds (cross-links) among individual peptides or proteins in the complex. 45–48 Tyrosines were also reported to facilitate Aβ cross-linking in the presence of copper and hydrogen peroxide. 49 Importantly, dityrosine cross-links were found in amyloid plaques from AD brain tissue and were proposed to play a central role in AD. 50 Because there is a single tyrosine (and no tryptophans) in the Aβ sequence (Y10), we here used a simple model of covalent cross-linking, in which covalent bonds (cross-links) occurred exclusively among tyrosines. PICUP/SDS-PAGE or CHICUP/SDS-PAGE experiments are typically performed by first

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dissolving Aβ in the buffer and then exposing the solution to the cross-linking reaction, which is typically short, ranging from several milliseconds to several seconds. Finally, the reaction is quenched to avoid peptide degradation due to radicals in the buffer. 13–15 We performed DMD4B-HYDRA simulations in two stages to mimic in vitro sample preparation and crosslinking process. In the first stage, 8 statistically independent trajectories, each 40 × 106 time units long, were acquired. Each trajectory contained 32 initially unstructured and spatially separated peptides that self-assembled during simulations without any cross-linking reaction. These simulations have been shown to result in oligomer size distributions consistent with PICUP/SDS-PAGE data. 21,26 This first stage of simulations mimics Aβ dynamics in the sample buffer during the experiment prior to cross-linking. In the second stage, two types of simulations were acquired, using the final configurations of the first stage of simulations as the initial configurations. Each of the two types of the second-stage simulations was performed using 8 statistically independent, 50 × 106 time units-long trajectories that contained 32 peptides each. The first type of simulations allowed for the cross-linking reaction to mimic either PICUP or CHICUP. The cross-linking reaction was implemented in the DMD4BHYDRA approach as a strong attractive interaction among proximate tyrosines as described in Methods. The resulting ensembles consisted of monomers and covalently cross-linked oligomers of different orders. The second type of simulations was performed in the absence of cross-linking and served as a control set of simulations. The cross-linking and control trajectories acquired in the second stage of simulations were then analyzed as described below in detail. We use the following abbreviations for the selected Aβ peptide regions: the N-terminal region D1-R5 (NTR), the central hydrophobic cluster L17-A21 (CHC), the mid-hydrophobic cluster I31-V36 (MHR), and the C-terminal region V39-V40 or V39-A42 (CTR) for Aβ40 or Aβ42 , respectively.

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Time Evolution of Cross-Linking The average number of covalent bonds among proximate tyrosines (YY bonds or cross-links) and the corresponding standard error were calculated during the second stage of simulations for both Aβ40 and Aβ42 (Fig. 1a, black and red curves, respectively). During the first 10 × 106 time units, the number of YY bonds in Aβ40 and Aβ42 increased rapidly with no obvious difference between the two alloforms. After 10 × 106 time units, the number of YY bonds appeared higher for Aβ40 than for Aβ42 , as reflected in a higher average number of YY bonds. At ∼ 45 × 106 time units, the number of YY bonds for both Aβ40 and Aβ42 reached a steady-state value as the average number of YY cross-links did not increase within the last ∼ 5 × 106 time units (Fig. 1a). To estimate the efficiency of YY cross-linking in simulations, we also calculated the number of tyrosines that did not contribute to covalent cross-linking and thus remained free (Fig. 1b). We observed that at the end of the second stage of simulations, there were on average still ∼9 and ∼12 (out of 32) free tyrosines in Aβ40 and Aβ42 ensembles, resulting in cross-linking efficiency of 72% and 63%, respectively. The above data demonstrated that Aβ40 tends to have higher cross-linking efficiency than Aβ42 . These estimates of cross-linking efficiency are somewhat below the experimentally reported Aβ cross-linking efficiency of ∼80%. 13

Solvent Exposure of Cross-Linked and Noncross-Linked Ensembles The above results show that Aβ40 was more efficient in facilitating cross-linking than Aβ42 . As cross-linking in our simulations was facilitated by tyrosine-tyrosine bonding alone, we here examined solvent exposure of tyrosines in Aβ40 and Aβ42 ensembles. We thus calculated the CG-SASA per amino acid for entire noncross-linked Aβ40 and Aβ42 ensembles at the end of stage 1 and cross-linked and noncross-linked Aβ40 and Aβ42 ensembles at the end of stage 2 of simulations. The results in Fig. 2 show that while the NTR and G9 were significantly more exposed to solvent in Aβ42 than in Aβ40 ensembles, H6 and the Y10-E11 region that included tyrosine were significantly more exposed to the solvent in noncross-linked Aβ40 than 13

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Figure 1: Time evolution of the number of (a) YY cross-linking bonds and (b) free tyrosines. For each time frame, the average number of YY bonds and the number of “free” tyrosines that did not participate in covalent bonding was calculated by averaging over the 8 trajectories. The error bars correspond to SEM values.

in noncross-linked Aβ42 simulations, both at the end of stage 1 (Fig. 2a) and stage 2 (Fig. 2b). The CG-SASA per amino acid in noncross-linked Aβ40 and Aβ42 ensembles at the end of stage 1 was indistinguishable from the CG-SASA per amino acid in noncross-linked Aβ40 and Aβ42 ensembles at the end of stage 2. Cross-linking significantly reduced the solvent exposure of the H6-Y10 and N27 in Aβ40 ensembles and the S8-Y10, Q15, and K28 in Aβ42 ensembles (Fig. 2c,d). Nonetheless, H6 and the Y10-E11 region, which includes tyrosine, remained more exposed to the solvent in cross-linked Aβ40 than in cross-linked Aβ42 ensembles (Fig. 2e). The increased solvent exposure of tyrosines in noncross-linked Aβ40 (relative to noncross-linked Aβ42 ) ensembles, which increases the probability of tyrosine-tyrosine interactions, provides an explanation of why cross-linking efficiency was higher in Aβ40 than in Aβ42 ensembles.

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Figure 2: CG-SASA per amino acid in noncross-linked and cross-linked Aβ40 and Aβ42 ensembles. CG-SASA per amino acid was calculated for each of the 32 peptides, regardless of the assembly state, and then averaged over all peptides, all frames (20−40×106 and 30 − 50 × 106 time units of stage 1 and stage 2 simulations, respectively; frames were 105 time units apart), and all trajectories. Comparison of CG-SASA per amino acid of noncrosslinked Aβ40 and Aβ42 ensembles after (a) stage 1 and (b) stage 2 simulations. CG-SASA per amino acid of noncross-linked and cross-linked (c) Aβ40 and (d) Aβ42 ensembles. (e) CGSASA per amino acid of cross-linked Aβ40 and Aβ42 ensembles. The error bars correspond to SEM values.

Aβ Assembly under Cross-Linking Conditions Unlike in control simulations, where oligomers formed exclusively through non-covalent bonding, in simulations that mimicked cross-linking, oligomers formed through non-covalent as well as covalent bonding. Time evolution of the resulting oligomer size distributions, calculated as described in Methods, is shown in Fig. 3 for both noncross-linked (control) and cross-linked Aβ40 and Aβ42 ensembles. As reported previously, by the end of the first stage of 15

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simulations, Aβ40 and Aβ42 oligomer size distributions reached a quasi-steady state. 26 This observation was further confirmed by inspection of time evolution of control Aβ40 and Aβ42 ensembles, which revealed only minor changes in the oligomer size distributions during the second stage of simulations (Fig. 3, black curves). In contrast, the ensembles subjected to cross-linking displayed significantly less stable oligomer size distributions, which was more pronounced in Aβ40 than in Aβ42 ensembles (Fig. 3, red curves). Further inspection revealed that in the cross-linked Aβ40 ensembles, peptides tended to collapse and form large oligomers. We observed a peak in the Aβ40 distribution at oligomer size of 32 (the maximum size of assembly in our simulations) that increased with time. At 50 × 106 time units, in 2 out of 8 trajectories all 32 Aβ40 peptides formed a single assembly, as shown in Fig. 4a. A similar albeit less obvious trend was observed in Aβ42 ensembles, which were characterized by a decrease of LMW oligomers at the expense of of larger oligomers, whereby the largest oligomers were 20-mers, as shown in Fig. 4b. These simulation results suggest that cross-linking through tyrosine should enhance formation of larger protofibril-like oligomers, in particular for Aβ40 ensembles.

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Aβ40 0.4

Aβ42 t=+10x10

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0.2 0 0.4

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6

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0 0.4

t=+30x10

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Oligomer Size Figure 3: The effect of cross-linking on Aβ40 and Aβ42 oligomer size distributions. The oligomer size distributions for noncross-linked and cross-linked Aβ40 and Aβ42 ensembles were calculated every 10×106 time units as described in Methods. The error bars correspond to the SEM values.

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Figure 4: Examples of the largest (a) Aβ40 and (b) Aβ42 oligomers observed in cross-linking simulations. The red and blue beads correspond to the N-terminal D1 and the C-terminal amino acids V40 (Aβ40 ) or A42 (Aβ42 ). Orange beads represent tyrosines (Y10).

Cross-Linking via YY Bond Produces Only Dimers, Trimers, and Tetramers In experimental studies, after the cross-linking reaction with PICUP or CHICUP is quenched, SDS-PAGE is used to measure the resulting oligomer size distribution of covalently bonded (cross-linked) oligomers. Because SDS is a detergent, it will disassemble non-covalently bonded peptides, which will migrate as monomers and contribute to the monomeric band on the gel. Only covalently bonded oligomers will remain bonded and will contribute to oligomeric bands on the gel. To mimic the SDS-PAGE experiment, we thus extracted only covalently bonded oligomers from the cross-linked Aβ40 and Aβ42 ensembles and calculated their numbers as described in Methods. Table 1 shows the number of Aβ40 and Aβ42 monomers, dimers, trimers, and tetramers obtained from 8 cross-linked trajectories between 30 × 106 and 50 × 106 time units. No larger cross-linked oligomers were found in these simulations, demonstrating that Aβ40 can form oligomers via YY cross-linking only 18

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up to including trimers, and Aβ42 can form oligomers up to including tetramers. Because cross-linked tetramers were only observed in cross-linked Aβ42 ensembles and were very rare (Table 1), we only structurally analyzed Aβ40 and Aβ42 monomers, dimers, and trimers derived from cross-linked ensembles. Note that monomers and cross-linked oligomers extracted from cross-linked ensembles of conformations may have been attached to other oligomers through non-covalent bonds and formed large assemblies, such as those shown in Fig. 4. Table 1: The number of monomers and YY-bonded oligomers. These numbers were derived from 8 cross-linking trajectories using time frames between 30 × 106 and 50 × 106 time units (105 time units apart), in total 200 frames per trajectory, whereby each frame contained 32 peptides.

[YY]Aβ40 [YY]Aβ42

n=1 17253 [53.1%] 21218 [60.9%]

n=2 11884 [36.5%] 11072 [31.8%]

n=3 3393 [10.4%] 2374 [6.8%]

n=4 0 [0%] 179 [0.5%]

We then conducted structural analysis on both cross-linked and control (noncross-linked) conformational ensembles. In each case, we used 8 trajectories between 30 × 106 and 50 × 106 time units to extract monomer and oligomer conformations. In the following, the results of this structural analysis are described, whereby cross-linked conformations are compared to the corresponding conformations obtained in control simulations.

Cross-Linking Decreases β-Strand and Increases Coil Content in Aβ Oligomers To examine the effect of cross-linking on secondary structure of Aβ oligomers, we calculated the average turn, β-strand, and coil content in monomers, dimers, and trimers derived from cross-linked simulations and compared it to the corresponding content in control (noncrosslinked) conformations (Fig. 5, compare solid and dashed curves). Interestingly, the secondary structure of monomers was affected by cross-linking, although there were no intrapeptide

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covalent bonds present. This is a consequence of the fact that the monomer population was derived not only from actual monomers but also from individual peptides that were non-covalently attached to other peptides or were part of a larger assembly. Whereas the turn content in Aβ40 monomers was slightly larger in the cross-linked ensemble than in the control ensemble, cross-linking did not affect the turn content in Aβ42 monomers (Fig. 5a). The β-strand content was significantly higher in the cross-linked Aβ42 monomer population than in control Aβ42 monomer population, whereas a reverse effect was observed for Aβ40 (Fig. 5b). Cross-linking increased the coil content in Aβ40 monomers, while decreasing the coil content in Aβ42 monomers (Fig. 5c). Cross-linking increased the turn content in Aβ40 and Aβ42 dimers and had an opposite or no effect on the turn content in Aβ40 and Aβ42 trimers, respectively (Fig. 5a). Importantly, cross-linking significantly decreased β-strand content and increased coil content in Aβ40 and Aβ42 dimers and trimers (Fig. 5b,c). We also calculated turn, β-strand, and coil propensities per amino acid for each assembly state for both cross-linked and control ensembles (Figs. S1-S3). These data demonstrate that the effect of cross-linking on the secondary structure was not local (in the vicinity of Y10) but affected the entire peptide. The β-strand propensity is extremely high in noncross-linked dimers for both Aβ40 and Aβ42 , though the peak of Aβ40 is not as high as that of Aβ42 . This graph is calculated using the results between 30 × 106 and 50 × 106 time units in the second stage of simulations, which corresponds to actual time frames between 70 × 106 and 90 × 106 time units. This is because the control simulations from the second stage were just an extension of simulations obtained in the first stage. A comparison of the above secondary structure content for control simulations to previously reported data obtained for time frames between 20 × 106 and 40 × 106 time units 26 revealed that the β-strand content in Aβ40 and Aβ42 dimers, tetramers, and hexamers notably increased over time, whereas trimers and pentamers were less affected (compare Fig. 5b, dashed curves, with Fig. 5a in Ref. 26 ). Notably, Aβ40 and Aβ42 dimers had the highest β-strand content (Fig. 5b, dashed curves). This results elucidate the

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importance of dimers in Aβ assembly and provide evidence of a slow structural conversion of Aβ oligomers into assemblies with increased β-strand content, possibly on the pathway to fibril formation. 0.55

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0.35

(c)

0.3 0.25 0.2

1

2

3 4 Oligomer Size

5

6

Figure 5: Average (a) turn, (b) β-strand, and (c) coil content in cross-linked and noncross-linked Aβ40 and Aβ42 ensembles. The secondary structure (turn, βstrand, and coil) was calculated for each amino acid in the conformation, average over the number of peptides in the oligomer, and finally averaged over the entire monomer or oligomer population. Error bars correspond to SEM values.

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Quaternary Structure of Cross-Linked Aβ Oligomers Is Dominated by the YY Contact To explore the effect of YY cross-linking on tertiary and quaternary structure, we derived intra- and intermolecular contact maps, which show the frequency of tertiary and quaternary contacts for all pairs of amino acids in an oligomer, normalized by the oligomer order. In the following, this normalized frequency is referred to as the contact strength. Note that the tertiary contact map contains information about the folding contacts, whereas quaternary contact map offers insight into the contacts that drive oligomerization. We calculated contact maps based on proximity of Cα and well as Cβ atoms. As YY bonding occurred through the Cβ atoms of the proximate tyrosines, we expected to observe a stronger effect of YY cross-linking on the contacts among Cβ atoms. Both types of intramolecular contact maps, based on Cα and Cβ distances, indicate that YY cross-linking did not strongly affect tertiary contacts in Aβ40 and Aβ42 monomers, dimers, and trimers (Figs. S4 and S5). In contrast, quaternary structure of Aβ oligomers was altered by cross-linking. Fig. 6 shows quaternary contacts between pairs of Cβ atoms for cross-linked and control Aβ40 and Aβ42 dimers and trimers. As expected, quaternary contacts between tyrosine pairs dominated the quaternary contact maps of cross-linked Aβ40 and Aβ42 dimers and trimers. Apart from the strong YY contact, there were significantly fewer quaternary contacts in cross-linked than in control oligomers. The inhibition of quaternary Cβ contacts was particularly strong for Aβ40 dimers, in which the two peptides were held together predominantly through YY cross-linking bonds, whereas the contacts involving CHC, MHR, and CTR, which dominated the quaternary contact maps of control Aβ40 and Aβ42 oligomers, were only marginal. Overall, the contacts between the NTR and the other peptide regions were strongly inhibited by cross-linking, more so in Aβ40 than in Aβ42 (Fig. 6, orange frames). The Cβ -Cβ contacts between the CHC and the other peptide regions also decreased in strength due to cross-linking (Fig. 6, red frames). A similar observation was made also for quaternary contacts among a combined MHR and CTR (Fig. 6, gray frames). 22

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Interestingly, whereas cross-linking inhibited quaternary V12 contacts in Aβ40 oligomers, it enhanced these contacts in Aβ42 oligomers (Fig. 6, black arrows). In comparison, crosslinking slightly decreased V24 contacts in both Aβ40 and Aβ42 oligomers, but the effect was stronger for Aβ40 oligomers (Fig. 6, red arrows). In analogy to Fig. 6, Fig. S6 shows quaternary contact maps calculated based on Cα -Cα proximity, which revealed similarly inhibited quaternary contacts (with the exception of the YY contact) due to cross-linking in Aβ40 and Aβ42 oligomers. In addition, quaternary Cα -Cα contact maps revealed parallel contacts among peptides in cross-linked Aβ40 and Aβ42 oligomers within the S8-V12 region, which were not present in control oligomers. These parallel contacts were the strongest for Aβ40 trimers, which exhibited parallel contacts within the extended E2-V12 region (Fig. S6).

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Figure 6: Quaternary Cβ -Cβ contact maps of cross-linked and noncross-linked Aβ40 and Aβ42 dimers and trimers. Intermolecular contact maps calculated based on the proximity between pairs of Cβ atoms. The average normalized contact frequency values are displayed in the upper left triangle and the corresponding SEM values (< 0.01) are shown in the lower right triangle of each contact map. Quaternary contacts involving NTR, CHC, and combined mid-hydrophobic/C-terminal regions are enclosed in red, orange, and gray frames, respectively. Black and red arrows point to quaternary contacts of V12 and V24, respectively. The Y10-Y10 contact propensities in cross-linked oligomers were 1 and 2 for dimers and trimers, respectively, thus exceeding the color scale range.

Increased Solvent Accessibility of Cross-Linked Aβ Oligomers To examine whether or not cross-linking affects the solvent exposure of Aβ oligomers, we calculated the average CG-SASA per residue for monomers, dimers, and trimers derived from cross-linked and control ensembles. As shown in Fig. 7, cross-linking affected the CGSASA per residue of Aβ40 and Aβ42 monomers along the entire sequence. This may appear

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unexpected because YY covalent bonds could form only intermolecularly, which should not have affected the monomer structure. It is important to note that the monomer population in cross-linked ensembles contained also peptides that were non-covalently bonded to other peptides, thereby altering their structure, including CG-SASA values. Upon closer inspection of the CG-SASA per residue in monomers, we noted that cross-linking affected a larger number of residues in Aβ42 than in Aβ40 . On average, hydrophobic residues exhibited slightly increased CG-SASA values in cross-linked monomers (e.g. A2, F4, V12, I31, the two Cterminal residues), whereas glycines tended to have slightly decreased CG-SASA values. Relative to the CG-SASA changes in monomers, cross-linking had a much stronger effect on the solvent exposure in Aβ40 and Aβ42 dimers and trimers. Cross-linked Aβ40 dimers and trimers had increased CG-SASA values in the N-terminal region D1-R5 as well as in the entire extended region V12-V40 that includes CHC, MHR, and CTR relative to the respective control values (Fig. 7, left panel). Similarly, cross-linked Aβ42 dimers and trimers displayed increased CG-SASA values in the N-terminal region D1-H6, at V12, and in the extended region L17-A42 with exception of K28, which was associated with decreased CG-SASA relative to control values (Fig. 7, right panel). Unlike cross-linked Aβ42 trimers, cross-linked Aβ42 dimers also exhibited an increased CG-SASA value at H14 (Fig. 7, right panel). In contrast, H6 and S8-Y10 in Aβ40 dimers, D7-Y10 in Aβ40 trimers, S8-Y10, Q15, and K28 in Aβ42 dimers, and D7-E11, Q15, and K28 in Aβ42 trimers were associated with decreased SASA values relative to controls. Overall, cross-linking strongly decreased solvent exposure of Y10 and a few neighboring amino acids, while increasing exposure of the vast majority of hydrophobic amino acids in Aβ40 and Aβ42 dimers and trimers to the solvent. The decreased solvent accessibility of Y10 and neighboring amino acids was a direct consequence of covalent bond formation by tyrosines. In control ensembles, tyrosine were not treated as hydrophobic residues, so they did not participate in non-covalent contact formation. Consequently, Y10 had a high CG-SASA in control simulations. In contrast, in cross-linked ensembles tyrosines that were covalently bonded to each other moved closer to

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the core of cross-linked oligomers and their CG-SASA correspondingly decreased. The above findings help explain why cross-linked ensembles contained larger oligomers than control ensembles (Fig. 3). Because the hydrophobic effect drives Aβ oligomerization, cross-linked oligomers with increased solvent exposure of hydrophobic residues promoted self-assembly through non-covalent effective hydrophobic interactions, resulting in formation of large oligomers that were not observed in control ensembles (Fig. 4). We then compared CG-SASA values per amino acid in cross-linked Aβ40 and Aβ42 dimers (Fig. S7a) and trimers (Fig. S7b). In comparison to cross-linked Aβ40 dimers, cross-linked Aβ42 dimers showed increased solvent accessibility predominantly within the N-terminal half of the sequence (A2, F4-R5, D7-G9, H14, A21, N27). In contrast, cross-linked Aβ40 dimers exhibited higher CG-SASA values than cross-linked Aβ42 dimers in the following regions: D1, E3, H6, Y10-V12, Q15-V18, F20, and most significantly in the extended hydrophobic C-terminal region K28-A42. The large difference between Aβ40 and Aβ42 CG-SASA values in the extended hydrophobic C-terminal region K28-A42 was not observed in cross-linked trimers. The Y10-V12 region, which contains the cross-linking site Y10, was more solvent exposed in cross-linked Aβ40 than in cross-linked Aβ42 oligomers, in particular in trimers. Because cross-linked dimers were the most abundant cross-linked oligomers, the strong exposure of the hydrophobic C-terminal region to the solvent in cross-linked Aβ40 (relative to Aβ42 ) dimers explains the occurrence of large assemblies in the cross-linked Aβ40 ensembles that surpassed in size the largest assemblies observed in cross-linked Aβ42 ensembles. This distinct effect of covalent cross-linking on Aβ40 and Aβ42 dimers stems from a competition between covalent Y10-Y10 bonding and formation of non-covalent hydrophobic contacts, where the hydrophobic C-terminus plays the dominant role. Because the C-terminus is more hydrophobic in Aβ42 than in Aβ40 , non-covalent hydrophobic contacts between C-termini can better compete with the covalent Y10-Y10 bonding in Aβ42 , leaving the C-terminus more protected from the solvent.

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Aβ40

Noncross-linked Cross-linked

Aβ42

n=1

1.2 0.9 0.6 0.3

2

CG-SASA [nm ]

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0

n=2

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n=3

1.2 0.9 0.6 0.3 0

0

5

10 15 20 25 30 35 40 0

5

10 15 20 25 30 35 40

Residues Figure 7: CG-SASA values per amino acid of cross-linked and noncross-linked Aβ40 and Aβ42 monomers, dimers, and trimers The CG-SASA values were calculated as described in Methods. Error bars correspond to SEM values.

Cross-Linking Broadens and Shifts Free Energy Landscapes of Aβ Conformations We examined the effect of cross-linking on the free energy landscape by calculating PMF of cross-linked and noncross-linked Aβ40 and Aβ42 monomers, dimers and trimers, using the distance between the Cα atom of D1 and the center of mass of the peptide (N-CM distance) and the sum of CG-SASA values over all hydrophobic residues (hydrophobic CG-SASA) as the two reaction coordinates (see Methods). As displayed in Fig. 8, cross-linked Aβ40 and Aβ42 oligomers were characterized on average by both a broader range and larger N-CM 27

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distances as well as larger hydrophobic CG-SASA values than their noncross-linked counterparts. Comparing the free energy landscapes of noncross-linked Aβ40 and Aβ42 conformations, we observed that the latter were characterized by N-CM distances and hydrophobic CG-SASA values that were on average shifted to larger values, as expected based on the previously published findings. 19,21,26–28 Cross-linking affected Aβ40 and Aβ42 monomers although cross-linking acted intermolecularly. This was a consequence of the fact that crosslinked ensembles of monomers contained also peptides that were non-covalently bonded to other peptides. As observed in Fig. 8, the free energy landscape of cross-linked monomer ensembles was more expanded and included conformations with larger N-CM distances and hydrophobic CG-SASA values than the free energy landscape of noncross-linked monomer ensembles. As expected, cross-linking strongly affected the free energy landscapes of Aβ40 and Aβ42 dimers and trimers by broadening the free energy landscapes along both reaction coordinates as well as increasing N-CM distances and hydrophobic CG-SASA values.

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Figure 8: PMF landscapes of cross-linked and noncross-linked Aβ40 and Aβ42 monomers, dimers, and trimers. The N-CM distance and hydrophobic CG-SASA were used as the two reaction coordinates. The representative conformations corresponding to the lowest free energy are also displayed. The red and blue beads correspond to the N-terminal D1 and the C-terminal V40 (Aβ40 ) or A42 (Aβ42 ). Orange beads represent tyrosines (Y10). The color scale is in units of kB T.

To better quantify the effect of cross-linking on the two reaction coordinates, we derived the corresponding one-dimensional distributions of N-CM distances and hydrophobic CGSASA values (Figs. S8 and S9). Mann-Whitney U-test 44 was applied to find out whether or not cross-linking altered the distribution of N-CM distances and hydrophobic CG-SASA 29

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values in a statistically significant way. The results confirmed that cross-linked ensembles of monomers, dimers, and trimers were indeed associated with distributions that differed from those derived for noncross-linked ensembles (Figs. S8 and S9). Although N-CM distance and hydrophobic SASA distributions appeared similar for cross-linked and noncross-linked monomers, the distributions for cross-linked monomers were shifted to higher values for with respect to those for noncross-linked monomers. A comparison of hydrophobic CG-SASA distributions in cross-linked versus noncross-linked dimers and trimers revealed quite distinct and minimally overlapping distributions. Similarly, N-CM distance distributions for crosslinked oligomers were strongly shifted to higher values relative to those for noncross-linked oligomers, for trimers even more so than for dimers. Representative Aβ40 and Aβ42 monomer, dimer, and trimer conformations observed in noncross-linked and cross-linked ensembles are shown in Fig. 8. Cross-linked Aβ40 monomers were more unfolded and had less β-strand content than noncross-linked Aβ40 monomers, consistent with a decrease of the average β-strand content upon cross-linking (Fig. 5b). The opposite effect was observed for cross-linked Aβ42 monomers, which had more β-strand content than noncross-linked counterparts, also in agreement with an increase of the average β-strand content upon cross-linking (Fig. 5b). As mentioned above, cross-linked monomer ensembles contained non-covalently bonded (self-assembled) structures. Thus, this alloformspecific difference was most likely related to an increased ability of Aβ42 relative to Aβ40 to form the β-sheet structure upon self-assembly. Noncross-linked Aβ40 dimers and trimers were quasi-spherical with a well-defined core and tyrosines that were evenly distributed across the surface. In contrast, cross-linked Aβ40 dimers and trimers did not have a well-defined core, were elongated, and had covalentlybonded tyrosines positioned close to the center of mass of the oligomer. Cross-linked Aβ40 dimer and trimer conformations that corresponded to the free energy minima were mostly dumbbell-shaped, whereby in cross-linked Aβ40 trimers the dumbbell shape was more asymmetric.

We observed similar differences between noncross-linked and cross-linked Aβ42

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trimers, where cross-linked Aβ42 trimers that corresponded to the dominant free energy minimum adopted mostly an asymmetric dumbbell shape. Noncross-linked Aβ42 dimers displayed larger conformational diversity than noncross-linked Aβ40 dimers with at least two distinct free energy minima. The free energy landscape of noncross-linked Aβ42 dimers was dominated by relatively compact quasi-spherical conformations. Cross-linked Aβ42 dimers also showed more conformational diversity than cross-linked Aβ40 dimers, as reflected in the three distinct albeit comparably populated free energy landscape minima. The minimum with the lowest N-CM distance and hydrophobic SASA values was populated by quasispherical cross-linked Aβ42 dimers. The central minimum corresponded to less compact cross-linked Aβ42 dimers, which still retained some core but were more elongated. The minimum with the highest hydrophobic CG-SASA values was populated by the most disordered and unfolded cross-linked Aβ42 dimer morphologies. The above observations of representative conformations support our findings that cross-linking distinctly affects Aβ40 and Aβ42 populations.

Conclusions Characterization of soluble, heterogeneous oligomers formed by amyloidogenic proteins associated with human disease is challenging. Bitan et al. used PICUP cross-linking method combined with SDS-PAGE to characterize oligomer size distributions of Aβ40 and Aβ42 , which are the two predominant Aβ alloforms in the brain that are hypothesized to trigger AD pathology. 12 A recent study reported that a cross-linking process similar to PICUP may occur in the brain in the presence of copper and hydrogen peroxide (CHICUP), stabilizing Aβ oligomers and prolonging their lifetimes. 14 Both PICUP and CHICUP were shown to stabilize Aβ40 and Aβ42 oligomer size distributions and strongly inhibit formation of large oligomers or fibrils. Because Aβ40 and Aβ42 oligomers cross-linked via CHICUP may be a physiologically relevant species that contributes to AD pathology, 14,19 it is important to

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characterize their structure. A series of computational studies examined oligomer formation of several Aβ variants using the implicit-solvent DMD4B-HYDRA approach, which predicted several aspects of Aβ oligomer formation in agreement with available experimental data. 21,26–28,32 Here, we expanded the DMD4B-HYDRA approach to allow for cross-linking via tyrosine and then examined the effect of YY cross-linking on Aβ40 and Aβ42 oligomer formation and structure. Our results show that cross-linking among tyrosines caused unstable Aβ40 and Aβ42 oligomer size distributions and produced only LMW cross-linked oligomers up to Aβ40 trimers and up to Aβ42 tetramers, which is inconsistent with experimental observations. 12,14 We discovered that the main reason for not observing cross-linked Aβ40 and Aβ42 oligomers larger than trimers and tetramers, respectively, was the volume exclusion arising from a process of more than four Aβ peptides attempting to form a penta- or hexa-tyrosine complex required for a fully covalently bonded pentamer or hexamer, respectively (see Fig. 9).

Figure 9: Representative cross-linked Aβ40 and Aβ42 oligomers. The red and blue beads correspond to the N-terminal D1 and the C-terminal V40 (Aβ40 ) or A42 (Aβ42 ). Orange beads represent tyrosines (Y10).

Structural analysis revealed that cross-linking significantly reduced the β-strand and increased the coil content in Aβ40 and Aβ42 dimers and trimers, i.e. the predominant crosslinked oligomers. The cross-linking via tyrosines did not strongly affect the tertiary struc32

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ture. In contract, the quaternary structure of cross-linked Aβ40 and Aβ42 dimers and trimers was dominated by the YY contact, which was absent from the Aβ40 and Aβ42 dimers and trimers derived from control simulations. This effect was particularly strong in cross-linked Aβ40 dimers, which were almost exclusively bonded through the intermolecular YY contact. Further analysis showed that cross-linking increased the overall solvent exposure in all cross-linked oligomers in the NTR, CHC, MHR, and CTR. As the CHC, MHR, and CTR are hydrophobic regions, cross-linking thus strongly exposed hydrophobic residues to the solvent, which explains why cross-linked ensembles contained larger assemblies than control ensembles. The effect of cross-linking on the solvent exposure was the strongest in Aβ40 dimers. The solvent exposure of hydrophobic residues at the C-terminus (G29-V40) was significantly higher in cross-linked Aβ40 dimers than in cross-linked Aβ42 dimers. As cross-linked dimers were the dominant cross-linked oligomers for both Aβ40 and Aβ42 , the difference in the exposure of hydrophobic residues to the solvent caused formation of larger assemblies in cross-linked Aβ40 ensembles relative to those observed in cross-linked Aβ42 ensembles. Free energy landscapes of Aβ40 and Aβ42 conformations were explored using two reaction coordinates, the N-CM distance and hydrophobic CG-SASA. Upon cross-linking, free energy landscapes of Aβ40 and Aβ42 oligomers expanded along both reaction coordinates and moved to larger N-CM distances and hydrophobic CG-SASA values, consistent with quaternary structure and CG-SASA changes. The control Aβ40 and Aβ42 dimers and trimers were quasi-spherical globular structures as reported previously. 21,26 In contrast, cross-linked Aβ40 and Aβ42 dimers and trimers were significantly more elongated and lacked a well-defined core. Cross-linked Aβ40 dimers adopted a symmetric dumbbell shape with the two tyrosines close to the center of mass, while in cross-linked Aβ40 trimers the dumbbell shape was asymmetrical. Although the free energy landscape of control Aβ42 dimers showed multiple minima, the one corresponding to the more compact quasi-spherical conformations with relatively low solvent exposure of hydrophobic residues dominated the conformational ensemble. In contrast, the free energy landscape of cross-linked Aβ42 dimers revealed three distinct yet comparably

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populated minima, one corresponding to compact globular conformations and the remaining two associated with more extended structures that differed from one another by the degree of solvent exposure of hydrophobic residues. The free energy landscape of cross-linked Aβ42 trimers was dominated by asymmetric dumbbell-like conformations akin to cross-linked Aβ40 trimer conformations. Our computational study of the effect of cross-linking on Aβ40 and Aβ42 assembly relies on a cross-linking model, in which only tyrosines are involved in covalent bond formation because there are no tryptophans in the Aβ sequence. This model is based on the reported mechanism of cross-linking, in which dityrosine formation plays the critical role. 46,47,49,50 Our findings demonstrate that cross-linking via tyrosines facilitates formation of large assemblies, in particular in Aβ40, which is not consistent with atomic force microscopy data showing that cross-linking stabilizes oligomeric assemblies, which do not considerably grow in size with the incubation time. 14 Moreover, in vitro Aβ40 and Aβ42 oligomer size distributions derived either by PICUP/SDS-PAGE 12 or CHICUP/SDS-PAGE 14 show the presence of Aβ40 tetramers as well as Aβ42 pentamers, hexamers, and dodecamers, which our simple model of cross-linking via tyrosines cannot reproduce as discussed above. Our findings suggest that the cross-linking mechanism, based exclusively on covalent bond formation via tyrosines is not sufficient to account for experimental findings. Thus, in addition to tyrosine, other amino acids in Aβ may contribute to covalent cross-linking. Indeed, Bitan reported that the cross-linking efficiency of Aβ40 and Aβ42 was reduced when tyrosine was substituted by phenylalanine from ∼ 80/(in Aβ42), but remained non-zero. 13 There are two additional plausible cross-linking processes that may contribute to covalent cross-linking. The first one involves covalent bond formation between tyrosine and lysine and was proposed by Fancy and Kodadek, who introduced PICUP as a cross-linking method for protein complexes. 47 The second one involves covalent cross-linking among histidines, which was reported by Liu M. et al. to occur in the presence of light and molecular oxygen. 51 Because each Aβ peptide contains two lysines and three histidines, one or both of these additional processes may

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potentially contribute to formation of larger cross-linked oligomers in a way that would be consistent with experimental findings. The first one may not be able to explain the non-zero cross-linking efficiency after tyrosine is replaced by phenylalanine, so the second process may be more likely. Based on the results of this study combined with previously reported findings on Aβ cross-linking, we predict that cross-linking via multiple amino acids will help explain the observed stabilization of oligomeric states as well as the observation of cross-linked Aβ40 and Aβ42 oligomers larger than trimers and tetramers, respectively. We are currently testing these predictions and will report our findings in future work.

Associated Content As supplementary of the results displayed above, we plotted the turn, β-strand, and coil propensities per residue, tertiary Cβ -Cβ contact maps, tertiary and quaternary Cα -Cα contact maps, probability distribution of CG-SASA and N-CM distance of cross-linked and noncrosslinked monomers, dimers, and trimers of Aβ40 and Aβ42 as well as comparison between Aβ40 and Aβ42 CG-SASA values of cross-linked dimers and trimers.

Acknowledgment This research is not supported by any external funding.

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