Elucidating the Role of Hydroxylated Phenylalanine in the Formation

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Elucidating the Role of Hydroxylated Phenylalanine in Formation and Structure of Cross-Linked A# Oligomers Shuting Zhang, Dillion M Fox, and Brigita Urbanc J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b12120 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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The Journal of Physical Chemistry

Elucidating the Role of Hydroxylated Phenylalanine in Formation and Structure of Cross-Linked Aβ Oligomers 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] Fax: (215) 895-5934

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Abstract Amyloid β-protein (Aβ) oligomers play a seminal role in Alzheimer’s disease (AD). Cross-linking (X-linking), which can be used determine Aβ oligomer size distributions experimentally, was reported to stabilize Aβ oligomers. Aβ oligomers X-linked in the presence of copper and hydrogen peroxide may represent the proximate neurotoxic species in AD. Our previous computational study demonstrated that X-linking of Aβ40 and Aβ42 oligomers via tyrosines alone cannot explain experimental findings. Here, we explore three plausible X-linking mechanisms, which involve, in addition to tyrosine, also: lysine (mechanism 1), histidine (mechanism 2), and hydroxylated phenylalanine (mechanism 3). By examining the effect of X-linking on oligomer size distributions, we show that only mechanism 3 is consistent with experimental data. Our findings provide important insights into the two-step X-linking via mechanism 3, which consists of a simple covalent bonding via tyrosines in the presence of hydroxylated phenylalanines, followed by covalent bonding among tyrosines and hydroxylated phenylalanines. Structural analysis of X-lined Aβ oligomers revealed increased solvent exposure at the N-terminal region, which was previously associated with increased oligomer toxicity. Our results elucidate a potentially important role of phenylalanine hydroxylation and increased toxicity of Aβ oligomers induced by X-linking.

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Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder which is the most common cause of dementia among the elderly. 1,2 It is characterized by intracellular neurofibrillary tangles, extracellular amyloid plaques, and massive neuronal loss. Amyloid β-protein (Aβ) is the main proteinaceous component of amyloid plaques. Aβ is produced through sequential cleavages of amyloid precursor protein (APP) by β- and γ-secretases. 3 While the original amyloid cascade hypothesis emphasized the neurotoxicity of insoluble Aβ fibrils in amyloid plaques, 4 revised amyloid cascade hypothesis posits that soluble, low molecular weight (LMW) oligomers of Aβ trigger the AD pathology. 5,6 The two predominant Aβ alloforms, Aβ40 and Aβ42 , are 40 and 42 amino acids long, respectively, whereby Aβ40 lacks the two C-terminal amino acids I41A42 that are present in Aβ42 . Despite this relatively small primary structure difference between the two alloforms, oligomers formed by Aβ42 are more toxic. 7–9 Therefore, the study on Aβ oligomer structures is of vital importance in understanding the mechanism by which Aβ oligomers trigger AD. Due to a disordered nature and relatively short lifetime of Aβ oligomers that typically exist as a heterogeneous mixture of monomers and oligomers of various sizes, classical experimental methods for protein structure characterization can not be applied. To date, no in vitro study has revealed a 3D structure of Aβ40 or Aβ42 oligomers. Cross-linking (X-linking) methods, such as photo-induced cross-linking of unmodified proteins (PICUP), which covalently bonds peptides, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which separates X-linked oligomers according to their sizes, can be used to determine the oligomer size distribution of Aβ, 10,11 and other intrinsically disordered proteins, such as α-synuclein. 12–17 Bitan et al. first introduced PICUP/SDS-PAGE method to reveal differences between Aβ40 and Aβ42 oligomer formation, 10,11 which were corroborated by studies using ion mobility combined with mass spectroscopy, which is a method independent of PICUP chemistry. 18,19 While PICUP requires ruthenium complex Ru(Bpy), ammonium persulfate (APS), and light to facilitate X-linking, Williams et al. recently introduced copper 3



and hydrogen peroxide-induced cross-linking of unmodified proteins (CHICUP), 20 a technique that requires only copper and hydrogen peroxide, both present in the AD brain, 21,22 to trigger the X-linking process. Both PICUP and CHICUP resulted in comparable Aβ40 and Aβ42 oligomer size distributions, suggesting a common X-linking mechanism. 20 Williams et al. also reported that X-linking stabilizes Aβ oligomers and inhibits their conversion into fibrils, which prolongs membrane disruption mediated by Aβ oligomers. If the CHICUP process occurs in the AD brain, Aβ oligomers X-linked by CHICUP may represent the proximate neurotoxic species in AD 23 and examining the effect of X-linking on Aβ oligomer formation and structure may provide important insights relevant to AD. Molecular dynamics (MD) offers a way to glimpse into early stages of oligomer formation and structure of full-length Aβ 24 as well as other intrinsically disordered proteins. Fully atomistic MD simulations of Aβ oligomer formation, which utilize explicit models of protein and solvent, are still time-consuming and thus impractical. A more efficient form of MD, discrete molecular dynamics (DMD), can be used to speed up the simulations, in particular when applied in combination with a coarse grained (CG) protein model and implicit solvent. DMD4B-HYDRA approach, 25 which combines DMD with a four-bead model of protein 26 and amino acid-specific hydropathic interactions 25,27 has been utilized to investigate the oligomer formation of Aβ40 and Aβ42 25,28 as well as their naturally occurring isoforms, 29–32 folding of mucin domains, 33 and oligomer formation of stefin B, a globular amyloidogenic protein. 34 A multiscale MD approach, in which DMD4B-HYDRA conformations of Aβ oligomers were converted into fully atomistic structures and then examined by explicit-solvent MD, was shown to efficiently sample the broad phase space of oligomer conformations, leading to characterization of fully atomistic details of Aβ40 and Aβ42 oligomers up to including pentamers in water. 35,36 We recently extended the DMD4B-HYDRA approach by implementing covalent bonds among tyrosines in order to study the effect of X-linking on Aβ40 and Aβ42 oligomer size distributions and oligomer structures. 37 Zhang et al. showed that no X-linked oligomers larger

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than Aβ40 trimers and Aβ42 tetramers were able to form 37 in contradiction to the experimental findings. 11,20 Moreover, X-linking induced formation of large Aβ40 and Aβ42 oligomers bonded through both non-covalent and covalent bonds, 37 which cannot be reconciled with the results of the experimental study of Williams et al., who reported that X-linking inhibits Aβ40 and Aβ42 fibril formation by stabilizing the oligomeric states. 20 Zhang et al. also reported that X-linking via tyrosines altered the shapes of oligomers from globular to dumbbell-like. 37 Detailed structural analysis revealed that volume exclusion was the limiting factor preventing formation of larger X-linked Aβ oligomers, leading to a conclusion that amino acids other than tyrosine are involved in X-linking. 37 The mechanism of X-linking that occurs under PICUP or CHICUP conditions is not well understood, so it is not immediately clear, which amino acids other than tyrosine might contribute to this process. In this paper, we employ DMD4B-HYDRA simulations to examine three possible mechanisms of Aβ40 and Aβ42 X-linking that go beyond the simple tyrosine-tyrosine (YY) covalent bond formation and involve (in addition to tyrosine) lysine (mechanism 1), histidine (mechanism 2), and hydroxylated phenylalanine (mechanism 3), and assess their ability to produce the oligomer size distributions consistent with experimental findings.

Methods DMD4B-HYDRA Simulations Discrete Molecular Dynamics. Molecular dynamics (MD) can be reduced to discrete molecular dynamics (DMD) by approximating the continuous interparticle potentials by one or multiple square wells. 38–40 As a method driven by collisions between pairs of particles, DMD does not require numerical integration. Particles move with constant velocities between two successive collisions. DMD can be faster than MD by several orders of magnitude when the density of the system is not too high.

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Four-Bead Protein Model with Backbone Hydrogen Bonding. In the four-bead protein model, 26 each amino acid is represented by four beads (three for glycine). The four beads correspond to four groups in amino acid: amide (N), α carbon (Cα ) and carbonyl (C) groups forming the backbone, and the side chain represented by a β carbon (Cβ ) bead. This CG model uses the smallest number of beads to reflect the chiral nature of an individual amino acid and the backbone peptide structure. The covalent bond lengths and the angles between bond pairs were determined by the known folded protein structures of ∼7,700 proteins from the Protein Data Bank (PDB). 41 Hydrogen bonds can form between bead Ni and Cj , the amide bead of amino acid i and the carbonyl bead of another amino acid j, respectively, when the two beads are within the range of [0.40 nm, 0.42 nm]. The unit of energy in our simulation is defined using the interaction strength of hydrogen bond EHB , which also dictates the unit of temperature EHB /kB . We performed our simulations at T = 0.13, which was reported to be a good estimate of physiological temperature, 30 corresponding to an estimate of the hydrogen bond energy of EHB = 4.7 kcal/mol. Amino Acid-Specific Interactions Due to Hydropathy. In DMD4B-HYDRA approach, the amino acid-specific interactions between Cβ beads are based on the phenomenological hydropathy scale by Kyte and Doolittle. 42 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. The amino acid-specific interaction between two side-chain beads occur only of the two amino acids are both hydrophilic or both hydrophobic, otherwise they only interact via volume exclusion. The hydropathic interaction is implemented with a single square well potential with interaction distance 0.75 nm. The absolute value of the effective hydropathic interaction strength is EHP = 0.3 as in previous studies. Implementation of X-linking. Zhang et al. modeled a covalent bond as a square-well potential between two Cβ beads of the corresponding amino acids with interaction strength EY Y = 20 and interactions range of [0.40 nm, 0.42 nm]. 37 Here, we expand the X-linking 6


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interaction beyond the tyrosine-tyrosine (YY) covalent bond formation to explore three possible mechanisms of Aβ X-linking: 1. tyrosine-lysine (YK) X-linking, 2. histidine-histidine (HH) X-linking, and 3. tyrosine-hydroxylated phenylalanine(YF’) and hydroxylated phenylalanine-hydroxylated phenylalanine(F’F’) X-linking, where F’ stands for hydroxylated phenylalanine. In mechanism 1, the X-linking reaction was allowed among tyrosines and between a tyrosine and a lysine. In mechanism 2, the X-linking reaction was allowed among tyrosines and among histidines but not between a tyrosine and a histidine. The interaction strengths of YK and HH covalent bonds were EY K = EHH = 20 and the interaction range was [0.40 nm, 0.42 nm], which matches the previously reported YY X-linking implementation. In mechanism 3, hydroxylated phenylalanines can form covalent bonds among themselves or with tyrosines. As discussed in results, only a small fraction of phenylalanines was reported to convert to tyrosines under oxidative stress. YF’ bond is assigned a lower interaction strength EY F ′ = 10. F’F’ bond is assigned an even lower strength, EF ′ F ′ = 5. Simulation Protocol. 8 trajectories of simulations were performed with 32 Aβ40 or 32 Aβ42 peptides in each trajectory enclosed in a cubic simulation box of 25 nm. Initial states were acquired by running short high-temperature DMD simulations for each trajectory, where there was no force field allowing interaction, resulting in 8 independent states, each having 32 spatially separated unstructured peptides. The 8 trajectories were acquired at constant volume and temperature T = 0.13, 30 which is an estimate of physiological temperature in the Berendsen thermostat. 43 According to the definition of temperature 21 mvx2 = 21 kB T , q the average velocity of beads can be calculated by vx = kBmT , where m is the unit mass in DMD simulations. The unit mass is defined from the mass of alanine m = 1/4MA = 7



3 × 10−26 kg and thus determines vx ≈ 300 m s−1 and ∆t = ∆x/vx = 10−11 /300 ≈ 0.03 ps. The simulations were performed in two stages in order to mimic the experimental procedures of sample preparation followed by X-linking. In stage 1 of 40×106 time units (approximately 1.2 µs), each of the 8 trajectories was subjected to simulations with no X-linking allowed. In stage 2 of 50×106 time units (approximately 1.5 µs), four types of simulations were conducted in parallel using the final conformations from stage 1 as the initial states. Three of them have X-linking implemented corresponding to the three possible X-linking mechanisms and the fourth one is a control group without X-linking. Implementation of stage 2 simulations for mechanism 3 was more involved than for mechanisms 1 and 2 and consisted of two steps. In the first step, which was 20 M simulation time units long, marked as stage 21, phenylalanines were altered from hydrophobic to neutral amino acids, referred to as F’ (hydroxylated phenylalanines), mimicking the conversion into tyrosines, while tyrosines (that were originally present at position 10 of Aβ sequences) were allowed to form covalent bonds. In the second step, which was 30 M simulation time units long, marked as stage 2-2, all tyrosines (Y) as well as hydroxylated phenylalanines (F’) were allowed to form covalent bonds among each other with interaction strengths described above.

Structural Analysis All the analysis was conducted using Virtual Molecular Dynamics (VMD) scripting tools. 44 Visualization of oligomer conformations was also done using VMD. Oligomer Size Distribution. In the first type of oligomer size distribution, we did not distinguish between covalent and non-covalent bonds. If the distance between any two beads in different peptides was ≤ 0.5 nm, these two peptides were considered to belong to the same oligomer. We calculated the oligomer size distributions every 10×106 time units during stage 2 of simulations. For oligomer size distributions at time T , oligomer size distribution at three time frames (T −106 , T −0.5×106 , and T ) were averaged for each trajectory. The average and

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SEM values were calculated using only the eight resulting statistically-independent oligomer size distributions. X-linked Oligomer Size Distributions. In addition to the first type of oligomer size distribution, we also calculated the X-linked oligomer size distributions, where only covalently bonded oligomers were considered. Time frames between 30 × 106 and 50 × 106 time units of stage 2 that were 105 time units apart (200 frames) were used to calculate the Xlinked oligomer size distributions for each trajectory for mechanisms 1 and 2. For mechanism 3 was implemented using two steps of stage 2 (2-1 and 2-2, respectively) simulations and frames between 15 × 106 and 20 × 106 of stage 2-1 and frames between 40 × 106 and 50 × 106 of stage 2-2 were used for this analysis. The average X-linked oligomer size distributions and the corresponding SEM values were calculated using the statistics of the 8 independent trajectories. Time Evolution of X-linking. The number of YY, YF, and FF covalent bonds was calculated during stage 2 of simulations, using time frames that were 105 time units apart. Intra- and interpeptide bonds were counted separately. At each time frame, the average number of covalent bonds and the standard error of the mean (SEM) were calculated from the 8 independent trajectories. Secondary Structure. The secondary structures derived using the STRIDE algorithm 45 was implemented in VMD. 44 In our analysis we calculated the propensities of the three predominant secondary structure: β-strand, turn, and coil. The average β-strand, turn, and coil propensities was calculated by averaging over all the peptides in the oligomer, then over all oligomers derived from X-linked oligomer size distributions, where the corresponding SEM values were also calculated.

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Contact Maps. The contact map is a matrix of contact frequencies of different pairs of amino acids normalized over all oligomers under study. When the distance between two Cβ atoms was ≤ 0.5 nm, the two amino acids are considered to be in contact. The normalized contact frequencies were displayed in the upper left triangle of the contact maps whereas the lower right triangle showed the corresponding SEM values. Tertiary (intrapeptide) and quaternary (interpeptide) contacts were distinguished. The number of tertiary and quaternary contacts was first averaged over all peptides in the oligomer, then averaged over all oligomers under study. The corresponding SEM values were also calculated. CG Solvent Accessible Surface Area (SASA). The calculation of SASA was performed within VMD. 44 For each atom (bead), a spherical surface 0.14 nm from its van der Waals surface was considered, and the area of this surface that did not overlap with the surface area of any other atoms contributed to the CG-SASA calculation. In addition to the four beads in the four-bead model the backbone carbonyl oxygen and amide hydrogen of each amino acid were also included. The CG-SASA of one amino acid was the sum of the ”free” surface areas of all atoms of this amino acid. The amino acid-specific CG-SASA was averaged over all peptides in the oligomer then averaged over all oligomers. The SEM values were also calculated from the statistics of all oligomers under study. Potential of Mean Force (PMF). We calculated the potential of mean force using the two-dimensional distribution of conformations with respect to two reaction coordinates: X1 , the distance between the Cα atoms of N-terminal amino acid and the center of mass of the oligomer (the N-CM distance) and X2 , the sum of CG-SASA over all hydrophobic amino acids where I, V, F, L, M, C, and A are considered hydrophobic (the hydrophobic CGSASA). We first calculated the N-CM distance and hydrophobic CG-SASA for each peptide and then averaged over all the peptides in the oligomer. From the calculated results of the two reaction coordinates, the two-dimensional distribution of conformations (with bin size of 0.1 nm × 0.3 nm2 ), P (X1 , X2 ), was calculated. Then the PMF values were calculated 10


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as βW (X1, X2 ) = − ln P (X1 , X2 ) + M, where M is a constant. We also calculated the corresponding one-dimensional distributions the N-CM distance, the hydrophobic CG-SASA and the distance between the Cα atoms of C-terminal amino acid (V40 Aβ40 and I42 Aβ42 ) and the center of mass (CM) of the oligomer (the C-CM distance). The representative conformations are conformations corresponding to the minimum of the PMFs.

Results and Discussion The primary structure of Aβ42 is: 1

DAEFRHDSGY

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EVHHQKLVFF

21

AEDVGSNKGA

31

IIGLMVGGVV

41

IA.

Aβ40 , which is two amino acids shorter, lacks the two C-terminal amino acids I41A42. In our previous study, we examined the effect of X-linking via tyrosines on Aβ40 and Aβ42 oligomer formation and resulting oligomer conformations. 37 Our results demonstrated that formation of covalent bonds among tyrosines alone is not consistent with two in vitro observations: (a) stabilization of LMW oligomers by X-linking 20 and (b) Aβ40 and Aβ42 oligomer size distributions (PICUP/SDS-PAGE or CHICUP/SDS-PAGE), which contain oligomers larger than trimers and tetramers, 11 respectively. We here examine three potential X-linking mechanisms, which involve in addition to YY X-linking also: 1. YK X-linking, 2. HH X-linking, 3. YF’ and F’F’ X-linking where F’ stands for hydroxylated phenylalanine. Mechanism 1 was proposed by Fancy and Kodadek, who argued that when a tyrosine forms a radical but there is no proximate tyrosine to bond to, it can form a covalent bond with a nearby lysine (K) or cysteine (C). 46 There are no cysteines in the Aβ sequence, therefore only YK X-linking is considered in addition

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to the YY X-linking in mechanism 1. In mechanism 2, we consider in addition to YY Xlinking also X-linking among histidines. This mechanism is based on the study by Liu et al., who reported that photo-oxidation of IgG1 antibody produced an His+32 intermediate, which X-linked with other histidines via a nucleophilic reaction. 47 Because of its nucleophilic nature, HH X-linking is distinct from the radical formation process during YY X-linking, therefore mechanism 2 does not involve X-linking between tyrosine and histidine. Mechanism 3 is based on a study by Ishimitsu et al. who showed that phenylalanines can be oxidized and converted to tyrosines in the presence of copper ions and hydrogen peroxide, which is consistent with CHICUP conditions. 48 Although only 5-15% of phenylalanines were oxidized and converted to tyrosines, this mechanism could occur under radical forming conditions similar to those used in PICUP or CHICUP. The implementation of mechanism 3 involves two consecutive steps: (i) hydroxylation of phenylalanine, F, into a tyrosine-like F’, followed by structural reorganization that allows for X-linking via tyrosines only (F’ is not involved in X-linking at this stage) and (ii) X-linking among Y and F’ residues, which results in formation of interpeptide YY covalent bonds as well as intra- and interpeptide YF’ and F’F’ covalent bonds. To account for a relatively low reaction yield, we use lower interaction strengths of the YF’ and F’F’ covalent bonds, as described in Methods. Each Aβ peptide has two lysines, three histidines, and three phenylalanines, so any one of the three mechanisms could bypass the volume exclusion limitation of the simple YY X-linking, which prevented formation of Xlinked Aβ40 and Aβ42 oligomers larger than trimers and tetramers, respectively. 37 Mechanism 1, however, cannot explain why the X-linking efficiency is decreased but does not vanish when tyrosine in the Aβ sequence is substituted by phenylalanine. 49 The simulations were performed in two stages that mimic the PICUP or CHICUP experimental process, which is the same as the already reported for simulations of the simple X-linking mechanism via tyrosines. 37 In the first, 40 M time units–long stage, peptides were allowed to assemble in the absence of X-linking. In the second, 50 M time units–long stage, four types of simulations were performed, three corresponding to each of the X-linking mech-

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anisms and one control type of simulations with no X-linking. The final conformations of the first stage were used as the initial conformations for all the four types of simulations. For each type of simulations of either stage, we acquired eight independent trajectories to feed the statistical analysis as described in Methods.

Assessment of the Three X-linking Mechanisms Aβ40 oligomer size distribution determined by PICUP/SDS-PAGE consisted of monomers through tetramers with abundances decreasing with oligomer order whereas Aβ42 oligomer size distribution was multimodal and displayed increased pentamer and hexamer abundances. 11 PICUP and CHICUP X-linking combined with SDS-PAGE resulted in very similar Aβ40 and Aβ42 oligomer size distributions. 20 Both PICUP and CHICUP were shown to stabilize Aβ40 and Aβ42 oligomers by inhibiting their conversion into fibrils as established by monitoring X-linked Aβ40 and Aβ42 samples by ThT fluorescence spectroscopy and AFM imaging. 20 Only Mechanism 3 Produces Stable Aβ40 and Aβ42 Oligomer Size Distributions To assess the three mechanisms with respect to their ability to stabilize Aβ40 and Aβ42 oligomer size distributions, we monitored time evolution of the Aβ40 and Aβ42 oligomer size distributions resulting from stage 2 simulations for each of the three X-linking mechanisms. In these oligomer size distributions we did not distinguish between covalent and non-covalent bonds, which mimics in vitro assembly of X-linked Aβ samples. Because AFM images of X-linked Aβ samples revealed oligomeric assemblies, which did not significantly increase in size with incubation time, 20 the correct X-linking mechanism should account for stable in silico oligomer size distributions for both Aβ40 and Aβ42 . Figs. 1, 2, and 3 show Aβ40 and Aβ42 oligomer size distributions during stage 2 simulations, which were subjected to X-linking mechanisms 1, 2, and 3, respectively (red curves). Each oligomer size distribution is compared to the simulation time-matched control oligomer size distribution (Figs. 1, 2, 13



and 3, black curves). Even at the shortest X-linking time of 10 M simulation time units, mechanisms 1 and 2 produced multimodal Aβ40 oligomer size distributions with increased pentamer through decamer abundances (mechanism 1) or septamer abundances (mechanism 2), respectively. Interestingly, Aβ42 oligomer size distributions at 10 M time units of Xlinking via mechanisms 1 and 2 were less affected. At longer X-linking simulation times, mechanism 1 produced Aβ40 oligomer size distributions that appeared to stabilize oligomer orders of 4, 9, 12-13, and 23, whereas the corresponding Aβ42 oligomer size distribution displayed instability, resulting in a steady shift toward larger oligomers, including 32-mers. Mechanism 2 produced unstable Aβ40 and Aβ42 oligomer size distributions at X-linking simulation times longer than 30 M time units. For mechanism 3, where the stage 2 simulations were performed in two parts, Aβ40 oligomer size distribution exhibited a shift toward larger oligomers between stages 2-1 and 2-2 (Fig. 3, left column, red curves) as well as relative to control Aβ40 oligomer size distributions (Fig. 3, left column, red versus black curves). Despite this shift, Aβ40 oligomer size distribution remained stable during X-linking via mechanism 3 and no large oligomers were observed. Aβ42 oligomer size distributions remained stable during both steps of stage 2 simulations (Fig. 3, right column, red curves) and were also almost indistinguishable from the respective control Aβ42 oligomer size distributions (Fig. 3, right column, red versus black curves). Of the three X-linking mechanisms, only mechanism 3 produced both stable Aβ40 and Aβ42 oligomers, which is consistent with previously reported PICUP and CHICUP stabilization of oligomers and inhibition of fibril formation. 20

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Aβ40 0.4

Aβ42 t=+10x10

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Oligomer Size Figure 1: Time evolution of Aβ40 and Aβ42 oligomer size distributions during mechanism 1 X-linking simulations. The oligomer size distributions of control (non-X-linked, black curves) and X-linked (red curves) Aβ40 and Aβ42 ensembles were calculated every 10 × 106 time units of stage 2 simulations as described in Methods. Error bars correspond to the SEM values.

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Aβ40 0.4

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0 0.4

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Aβ40 0.4

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t=+10x10 (YY)


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t=+20x10 (YY)

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0 0.4

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t=+30x10 (YY/YF’/F’F’)

0.2 0 0.4

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t=+40x10 (YY/YF’/F’F’)

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t=+50x10 (YY/YF’/F’F’)

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Only Mechanism 3 Captures the Multimodal Character of X-Linked Aβ42 Oligomer Size Distribution Stage 2 simulations were designed to mimic the X-linking reaction in the in vitro Aβ samples prior to gel electrophoresis. We calculated Aβ40 and Aβ42 oligomer size distributions of only X-linked oligomers, referred hereafter as X-linked oligomer size distributions, in which each oligomer is comprised of covalently bonded peptide, as described in Methods. The X-linked oligomer size distribution mimics the effect of SDS during gel electrophoresis, which disassembles oligomers into monomers unless they are covalently bonded. The correct X-linking

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mechanism is expected to capture Aβ40 and Aβ42 oligomer size distributions as determined by PICUP/SDS-PAGE or CHICUP/SDS-PAGE. X-linked Aβ40 and Aβ42 oligomer size distributions were calculated using time frames between 30-50 M simulation time units and compared with the oligomer size distributions of non-X-linked (control) groups within the same time interval, as described in Methods. The results for all three X-linking mechanisms are displayed in Figs. 4- 5. A comparison between X-linked Aβ40 and Aβ42 oligomer size distributions is displayed in Fig. S1. Whereas mechanism 1 resulted in a slightly increased abundance of X-linked pentamers of Aβ42 relative to Aβ40 (Fig. S1a), mechanism 2 resulted in more X-linked Aβ40 than Aβ42 oligomers, primarily dimers (Fig. S1b). Importantly, both mechanism 1 and 2 produced X-linked Aβ40 and Aβ42 oligomer size distributions with monomer and oligomer abundances monotonically decreasing with oligomer order. Whereas such dependence is consistent with in vitro Aβ40 oligomer size distribution, it is not consistent with the multimodal character of the in vitro Aβ42 oligomer size distribution. 11,20 Stage 2-1 of X-linking via mechanism 3, where only tyrosines were allowed to form covalent bonds (and phenylalanines were hydroxylated), produced X-linked oligomers no larger than tetramers for both Aβ40 and Aβ42 , which is consistent with our previous findings that Xlinking via tyrosines alone is unable to produce large X-linked oligomers. 37 During stage 2-2 of X-linking via mechanism 3, the propensities of both X-linked Aβ40 and Aβ42 monomers and dimers obviously decreased and larger X-linked oligomers, such as pentamers and hexamers, were able to form. Stage 2-2 of X-linking via mechanism 3 resulted in X-linked Aβ40 oligomer size distribution with monomer and oligomer abundances monotonically decreasing with oligomer order and X-linked Aβ42 oligomer size distribution with a bimodal character, i.e. increased monomer and hexamer abundances, which is, given sample size limitations in computer simulations, qualitatively consistent with in vitro Aβ40 and Aβ42 oligomer size distributions, respectively. Of the three mechanisms explores in this study, mechanism 3 produced the most distinct X-linked Aβ40 and Aβ42 oligomer size distributions (Fig. S1c), in accord with experimental observations. 11,20

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Aβ40

Aβ42

Mechanism 1: YY/YK 0.5 0.4 0.3 0.2

Probability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.1 0

Mechanism 2: YY/HH 0.5 0.4 0.3 0.2 0.1 0

0

5

10

15

0

5

10

15

20

Oligomer Size Figure 4: The X-linked oligomer size distributions of Aβ40 and Aβ42 compared to non-X-linked distributions for mechanisms 1 and 2. The X-linked oligomer size distributions were calculated and averaged as described in Methods. Error bars correspond to SEM values.

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Aβ40

0.7

Aβ42

Mechanism 3: Stage 2-1 (F to F’ & YY)

0.6 0.5 0.4 0.3 0.2

Probability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.1 0

Mechanism 3: Stage 2-2 (YY/YF’/F’F’)

0.6 0.5 0.4 0.3 0.2 0.1 0

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15

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20

Oligomer Size Figure 5: The X-linked oligomer size distributions of Aβ40 and Aβ42 compared to non-X-linked distributions for mechanism 3. The X-linked oligomer size distributions were calculated and averaged as described in Methods. Error bars correspond to SEM values.

X-Linking Affects Distance Distributions Between Amino Acids Involved in Covalent Bond Formation It is important to consider that all three X-linking mechanisms included the simple X-linking via tyrosines, which was reported in our previous study to induce profound changes in Xlinked oligomers. 37 The similarity of the X-linked oligomer size distributions obtained via mechanism 3 to the non-X-linked oligomer size distributions is thus an indication that the additional covalent bonds (YF’ and F’F’) that formed during X-linking stage 2-2 simulations

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countered the conformational changes induced by simple X-linking via tyrosines alone. To explicitly demonstrate that covalent bonds successfully formed during stage 2 simulations for all three X-linking mechanisms, we calculated normalized distance distributions between the Cβ atoms of amino acids involved in X-linking: YY and YK (mechanism 1), YY and HH (mechanism 2), and YY, YF’, and F’F’ (mechanism 3) for the most abundant X-linked Aβ40 and Aβ42 oligomers: dimers and trimers (mechanisms 1 and 2) and dimers, trimers, and hexamers (mechanism 3). The results, shown in Figs. 6 (mechanism 1), 7 (mechanism 2), and 8, 9 (mechanism 3) as red curves, were compared to the corresponding distance distributions calculated from stage 2 simulations of non-X-linked Aβ40 and Aβ42 oligomers. Whereas X-linking via tyrosines induced formation of interpeptide covalent bonds exclusively, X-linking via mechanisms 1-3 resulted in formation of intra- and interpeptide covalent bonds. Our results on Figs. 6, 7, 8, and 9 show distance distributions for both types of covalent bonds, excluding intrapeptide covalent bonds that formed due to proximity of the adjacent amino acids in Aβ sequences, H13-H14 and F19-F20. Unlike the distance distributions corresponding to non-X-linked oligomers, all distance distributions within X-linked oligomers exhibited a strong peak between 4.0 ˚ A and 4.2 ˚ A, reflecting formation of covalent bonds. This peak in the distance distributions of X-linked ensembles decreased with oligomer order for all the three mechanisms, indicating a less efficient X-linking with increased oligomer order. In contrast to intrapeptide X-linking, which affected the tertiary structure of resulting conformations, interpeptide X-linking contributed to formation of Xlinked oligomers. Because X-linking via mechanisms 1 and 2 involved a charged hydrophilic (K) and hydrophilic (H) amino acids in addition to hydrophilic Y, covalent bonding among hydrophilic amino acids, which were exposed to the solvent in non-X-linked conformations, increased solvent exposure of hydrophobic amino acids, which facilitated formation of unstable oligomer size distributions, similar to the effect of simple X-linking via tyrosines. 37 X-linking via mechanisms 1 and 2 facilitated formation of X-linked oligomers through interpeptide YY and YK (mechanism 1) or YY and HH (mechanism 2) covalent bonding. The

21



peaks in the YY and YK distance distributions (between 4.0 ˚ A and 4.2 ˚ A) corresponding to X-linking via mechanism 1 were of comparable heights, indicating that both types of covalent bonding contributed equally to X-linked oligomer formation. The peak corresponding to interpeptide YY covalent bonding in mechanism 2 was significantly higher than the peak associated with the interpeptide HH covalent bonding, demonstrating that interpeptide YY covalent bonding dominated X-linked oligomer formation. In X-linking via mechanism 3, in stage 2-1 only Y was allowed to form covalent bonds, whereas in stage 2-2 both Y and F’ were involved in covalent bond formation. Because interpeptide YY covalent bonds were allowed to form earlier than YF’ and F’F’ covalent bonds, the YY covalent bonds were more abundant than interpeptide YF’ and interpeptide F’F’ bonds as reflected in the a higher peak in distance distributions (Fig. 9). This difference decreased with oligomer order, reflecting the increasing importance of F’ (relative to Y) in formation of larger X-linked oligomers, such as hexamers, during stage 2-2.

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Figure 6: The effect of X-linking via mechanism 1 on YY and YK distance distributions. YY and YK distance distributions in X-linked Aβ40 and Aβ42 dimers and trimers were obtained from the last 20 × 106 time units of stage 2 X-linking simulations via mechanism 1. In each oligomer, distances between all YY and YK pairs were extracted and the corresponding normalized distributions were calculated.

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Figure 7: The effect of X-linking via mechanism 2 on YY and HH distance distributions. YY and HH distance distributions in X-linked Aβ40 and Aβ42 dimers and trimers were obtained from the last 20 × 106 time units of stage 2 X-linking simulations via mechanism 2. In each oligomer, distances between all YY and HH pairs were extracted and the corresponding normalized distributions were calculated.

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Figure 8: The effect of X-linking via mechanism 3 on intrapeptide YF/YF’ and FF/F’F’ distance distributions. Intrapeptide YF/YF’ and FF/F’F’ distance distributions are shown for control and X-linked Aβ40 and Aβ42 dimers, trimers, and hexamers. X-linked oligomers were extracted from the last 10 × 106 time units of stage 2-2 X-linking simulations via mechanism 3. In each oligomer, distances between all YF (control) or YF’ (X-linked) and FF (control) or F’F’ (X-linked) pairs were extracted and the corresponding normalized distributions were calculated.

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Figure 9: The effect of X-linking via mechanism 3 on interpeptide YY, YF/YF’, and FF/F’F’ distance distributions. Interpeptide YY, YF/YF’, and FF/F’F’ distance distributions are shown for control and X-linked Aβ40 and Aβ42 dimers, trimers, and hexamers. X-linked oligomers were extracted from the last 10 × 106 time units of stage 2-2 X-linking simulations via mechanism 3. In each oligomer, distances between all YY, YF (control) or YF’ (X-linked), and FF (control) or F’F’ (X-linked) pairs were extracted and the corresponding normalized distributions were calculated.

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Structural Analysis of Oligomers X-Linked Via Mechanism 3 Our comparison of three X-linking mechanisms revealed that of the three mechanisms under investigation, only mechanism 3 produced Aβ40 and Aβ42 oligomer size distributions consistent with experimental data. We then examined the effect of X-linking via mechanism 3 on resulting X-linked Aβ40 and Aβ42 oligomer conformations. Ensembles of Aβ40 and Aβ42 conformations subjected to two steps of X-linking (stage 2 simulations) via mechanism 3 reached quasi steady-state in the last 10 M simulation time units, which is reflected in the time evolution of the number of covalent bonds (Fig. S2). Unlike X-linking via tyrosines alone, which only gives rise to interpeptide covalent bonds, 37 X-linking via mechanism 3 induced formation of both intra- and interpeptide covalent bonds. Fig. S2 shows all five types of intrapeptide (YF’ and F’F’) and interpeptide (YY, YF’, and F’F’) covalent bonds that formed during the X-linking stage of simulations. The average number of interpeptide YY bonds increased rapidly during stage 2-1 with Aβ42 forming more YY bonds than Aβ40 , which is consistent with our previous findings 37 (Fig. S4a, left of the dashed line). During stage 2-2, where YF’ and F’F’ bonds were allowed to form, the number of YY bonds stopped increasing and reached a steady state (Fig. S4a, right of the dashed line). The number of YF’ and F’F’ bonds, both intra- and interpeptide, during stage 2-2 initially increased and reached a steady state within the last 10 M simulation time units (Fig. S4b-e). Whereas Aβ40 formed more intrapeptide YF’ and F’F’ bonds than Aβ42 (Fig. S4b,d), both peptides formed comparable number of interpeptide YF’ and F’F’ bonds. The above results provide insights into distinct X-linking processes occurring during stages 2-1 and 2-2 and highlight alloform specificity in covalent bond formation during X-linking via mechanism 3. In the following, we present structural analysis of X-linked monomers and oligomers extracted from stage 2 trajectories subjected to X-linking via mechanism 3 using time frames from the range of 40-50 M simulation time units, which corresponded to the last 10 M simulation time units of stage 2-2 X-linking. We further compare X-linked Aβ40 and Aβ42 oligomer conformations to oligomer conformations derived from the matching control trajec27



tories. The following abbreviations for the selected regions of Aβ sequences will be used: the N-terminal region D1-R5 (NTR), the central hydrophobic cluster L17-A21 (CHC), the midhydrophobic cluster I31-V36 (MHR), and the C-terminal region (CTR) V39-V40 or V39-A42 for Aβ40 or Aβ42 , respectively. X-linking Decreases β-Strand Content in Aβ Oligomers We calculated the average turn, β-strand, and coil content over all amino acids in X-linked oligomers and compared the resulting values with the corresponding secondary structure content in non-X-linked Aβ40 and Aβ42 oligomers (Fig. 10, solid and dashed curves). In comparison to non-X-linked oligomers, X-linked Aβ40 and Aβ42 oligomers had significantly lower β-strand content (Fig. 10b). All X-linked oligomers also had higher coil and turn content in comparison to non-X-linked oligomers. X-linked Aβ40 oligomers had higher turn content than X-linked Aβ42 oligomers except in trimers. X-linked Aβ40 and Aβ42 oligomers had comparable β-strand content in monomers. Whereas X-linked Aβ40 dimers and trimers had higher β-strand content than the corresponding Aβ42 oligomers, X-linked Aβ42 tetramers, pentamers, and hexamers had higher β-strand content than the corresponding Aβ40 oligomers. X-linked Aβ42 conformations had higher coil content than X-linked Aβ40 conformations for all oligomer sizes. Overall, X-linking via mechanism 3 altered the secondary structure of Aβ40 and Aβ42 conformations but the changes were significantly smaller than in the case of the simple X-linking via tyrosines. 37

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β-Strand Propensity

0.6

(a)

X-linked Aβ40 X-linked Aβ42 Non-X-linked Aβ40 Non-X-linked Aβ42

0.5 0.4 0.3 (b)

0.2

0.1 0.4 (c)

Coil Propensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Turn Propensity

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0.3

0.2

1

2

3 4 Oligomer Size

5

6

Figure 10: Average (a) turn, (b) β-strand, and (c) coil content in X-linked and non-X-linked Aβ40 and Aβ42 oligomers. Error bars correspond to SEM values.

We also calculated the amino acid-specific turn, β-strand, and coil propensities in Xlinked oligomers and compared them to propensities of the matching control oligomers. As shown in Figs. S3-S5, the effect of X-linking via mechanism 3 on the secondary structure was both alloform- and peptide region-specific. Overall, turn propensities increased in the E3-D7 region and decreased at Q15 in all X-linked Aβ40 and Aβ42 oligomers (Fig. S3). Increased turn propensities was observed in the Y10-V12 region for X-linked Aβ40 dimers and trimers. While increased turn propensities were observed at F’19 in all X-linked oligomers, the change in turn content at F’20 varied with oligomer size and alloform. The remaining changes in turn propensities due to X-linking were all alloform and oligomer size-specific. The turn 29



propensities in the E22-V24 region decreased in X-linked Aβ40 dimers and trimers and in Aβ42 hexamers. The L34-G38 region showed increased turn content for Aβ40 and Aβ42 dimers but was not strongly altered in trimers. Increased turn content was also observed in the L34-M35 of Aβ40 hexamers and A30-G37 of Aβ42 hexamers. Overall, X-linking via mechanism 3 decreased β-strand propensities in all X-linked oligomers (Fig. S4). X-linked Aβ42 oligomers were affected significantly more than Aβ40 oligomers, whereby in X-linked Aβ42 dimers almost all amino acid displayed decreased β-strand propensities. In X-linked Aβ42 trimers and hexamers, the largest decrease in β-strand propensities occurred in the R5-D7 and G9-H13 regions. In X-linked Aβ40 oligomers, the largest decrease in β-strand propensities was observed in the A2-F’4 region. The effect of X-linking on coil content also varied with alloform, oligomer order, and peptide regions (Fig. S5). Interestingly, X-linking increased coil propensities in the peptide region around Y10 in all Aβ42 oligomers but not in Aβ40 oligomers. X-linking increased coil propensities in the A2-F’4 region of all X-linked Aβ40 oligomers, however, the corresponding region (F’4 and neighboring amino acids) displayed decreased coil propensities in all X-linked Aβ42 oligomers. X-Linking Inhibits Tertiary Contacts between the NTR and CTR We calculated tertiary and quaternary contact maps between pairs of Cβ atoms for monomers, dimers, trimers, and hexamers of X-linked Aβ40 and Aβ42 (Fig. 11 and 12). Intrapeptide (tertiary) contacts provide information about the tertiary fold of individual peptide within the oligomer whereas interpeptide (quaternary) contacts elucidate peptide regions involved in oligomer self-assembly. Unlike simple X-linking via tyrosines, which did not alter tertiary structure of X-linked oligomers, 37 X-linking via mechanism 3 was expected to affect both tertiary and quaternary structure due to formation of intrapeptide YF’ and F’F’ covalent bonds. Fig. 11 shows that X-linking via mechanism 3 altered tertiary structure of Aβ40 and Aβ42 oligomers in two ways. First, the propensity (strength) of contacts formed by Y and F’ (F’4-Y10, F’4-F’19, F’4-F’20, Y10-F’19, Y10-F’20, and F’19-F’20) strongly in-

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creased in all X-linked oligomers with respect to the corresponding non-X-linked oligomers. These contacts corresponded to intrapeptide covalent bonds, which formed during stage 2-2 simulations. Concomitantly, the non-covalent tertiary contacts between the NTR (which includes F’4) and the remaining peptide regions, including the CTR were strongly inhibited (Fig. 11, red rectangles). Similarly, non-covalent contacts between the F’19-F’20 region and the remaining peptide regions almost disappeared (Fig. 11, black arrows). The inhibition of these non-covalent tertiary contacts resulted from the hydrophilic character of F’ as well as from the involvement of F’ in covalent bond formation, which favored covalent bond over non-covalent bond interactions.

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Figure 11: Tertiary Cβ -Cβ contact maps of non-X-linked and X-linked Aβ40 and Aβ42 dimers, trimers, and hexamers. Interpeptide 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. Tertiary contacts between CTR and the rest of the peptide sequence are enclosed in red frames. Tertiary contacts between F’19-F’20 and the rest of the peptide sequence are marked by black arrows.

X-Linking Strengthens Quaternary Structure of Aβ Oligomers at the C-Terminus Quaternary contacts highlight the peptide regions involved in oligomer formation. As shown in Fig. 12, X-linking altered the quaternary structure by inducing strong quaternary contacts between pairs of amino acids involved in X-linking (F’4-F’4, F’4-Y10, F’4-F’19, F’4-F’20,

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Y10-Y10, Y10-F’19, Y10-F’20, F’19-F’19, F’19-F’20, and F’20-F’20). The F’19-F’19, F’19F’20, and F’20-F’20 contact frequencies increased with oligomer size, which demonstrates that F’ played an important role in formation of larger X-linked oligomers during stage 2-2 simulations. Analogous to the changes in tertiary structure, X-linking also resulted in fewer and/or less frequent contacts of the NTR with the remaining sequence. In addition, due to the lack of hydrophobic character, the F’19-F’20 region formed fewer contacts with the rest of the sequence (Fig. 12, black arrows). X-linking induced increased number and frequency of parallel quaternary contacts within the NTR and also in the proximity of Y10, in particular in X-linked hexamers of both peptides. In the absence of X-linking, Aβ42 oligomer formation was driven primarily by contacts between the CTR and CHC regions as well as the contacts among the CTRs, as previously reported. 25 When Aβ42 ensembles were subjected to X-linking, the contacts within the CHC were inhibited except for those involved in covalent bonding: F’19-F’19, F’19-F’20, and F’20-F’20 (Fig. 12, red triangles). In contrast, X-linking induced stronger contacts among the I31-V40 region of Aβ40 and the I31-A42 region of Aβ42 (Fig. 12, orange triangles). Therefore, X-linking via mechanism 3 significantly altered the quaternary structure of both Aβ40 and Aβ42 oligomers. In addition to inducing new quaternary contacts among amino acids involved in X-linking, X-linking inhibited the long-range quaternary contacts of the NTR with both MHR and CTR, while increased the stability of quaternary contacts within CTR, MHR, and between MHR and CTR.

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Figure 12: Quaternary Cβ -Cβ contact maps of X-linked and non-X-linked Aβ40 and Aβ42 dimers and trimers. Interpeptide 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 among CHC regions are enclosed in red triangles. Quaternary contacts between pairs of I31-V40 or I31-A42 regions are enclosed in orange triangles. Black arrows point to quaternary contacts of F’19 and F’20 with other amino acids.

X-Linking Increases Solvent Exposure of the NTR and Its Distance from the Center of Mass in Aβ Oligomers Amino acid-specific CG solvent accessible surface area (CG-SASA) and distance to the center of mass (CM) was calculated for X-linked Aβ40 and Aβ42 conformations and compared to 34


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non-X-linked conformations as described in Methods (Figs. 13 and 14). These two quantities provide complementary structural information: CG-SASA quantifies the solvent exposure of each amino acid and the distance to the CM describes the arrangement of individual amino acids with respect to the CM of a conformation. Non-X-Linked X-Linked

Aβ40 1.5

Aβ42

n=1

1 0.5 0 1.5 2

CG-SASA [nm ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


n=2

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Residues Figure 13: Per residue CG-SASA values of non-X-linked and X-linked Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers. Error bars correspond to SEM values.

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Aβ40

Aβ42

n=1

3 2

Distance to Center of Mass [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 0 3

n=2

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40

Residues Figure 14: Distance from each amino acid to the CM for X-linked and control Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers. Error bars correspond to the SEM values.

X-linking via mechanism 3 had a significant effect on per-residue CG-SASA along the entire Aβ sequence, although the changes were smaller than in the case of simple X-linking via tyrosines. 37 The largest effect of X-linking was a sharp decrease in CG-SASA values in the S8Y10 region of all X-linked Aβ40 and Aβ42 oligomers, highlighting the importance of tyrosine in X-linking. In contrast, F’4, which was also involved in X-linking, and the neighboring residues became more exposed to the solvent. Similarly, the F’19-F’20 region and neighboring residues also exhibited increased solvent exposure. Consequently, X-linking via mechanism 3 increased solvent exposure at the NTR and CHC due to the lack of hydrophobic character 36


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of F’. The effect of X-linking on the solvent exposure of the CHC was stronger in dimers and trimers than in hexamers in both alloforms. In addition to the regions that included amino acids involved in X-linking, X-linking affected CG-SASA of other, more distant regions. Specifically, the A30-V40 and A30-A42 regions of Aβ40 and Aβ42 , respectively, became overall more exposed to the solvent in dimers and trimers of both peptide. The CG-SASA in this region also slightly increased in Aβ40 hexamers but was only marginally affected in Aβ42 hexamers. X-linking induced an increase in CG-SASA or some hydrophobic amino acids: V12 (in Aβ40 dimers, trimers, and hexamers as well as in Aβ42 dimers and hexamers) and V24 (in Aβ40 dimers and trimers, and hexamers as well as in Aβ42 dimers and trimers). The other changes in CG-SASA due to X-linking (e.g. H13-Q15 and S26-K28) were relatively minor and depended on oligomer order and alloform. The average distance of each amino acid to the CM within X-linked Aβ dimers, trimers, and hexamers displayed overall similar tendencies as the CG-SASA (Fig. 14). Y10 and its neighboring amino acids moved closer to the CM upon X-linking via mechanism 3. The NTR was driven farther from the CM. The F’19-F’20 region also exhibited increased distances to the CM except in Aβ40 hexamers. The MHR and CTR were was less affected by X-linking than the remaining sequence and even displayed decreased distances from the CM in Aβ40 hexamers. Unlike the simple X-linking via tyrosines, which caused Y10 and the neighboring amino acids to move closer to the CM and drove all other amino acids farther away from the CM (Fig. S6), X-linking via mechanism 3 had a weaker effect on the overall arrangement of amino acids within Aβ oligomers. X-linking Increases Polymorphism of Aβ Conformations To examine the effect of X-linking on free energy landscapes of Aβ oligomers, we calculated PMFs of X-linked and non-X-linked Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers using two reaction coordinates: the N-CM distance and the hydrophobic CGSASA as described in Methods (Fig. 15). The N-CM distance was selected because previous

37



DMD4B-HYDRA studies of Aβ40 and Aβ42 oligomers revealed distinct alloform-specific NTR conformations 23 and the hydrophobic CG-SASA quantifies the ability of the conformation to shield hydrophobic amino acids from the solvent.

Figure 15: PMF landscapes of non-X-linked and X-linked Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers. 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 (Y) and grey beads represent phenylalanines (F). The color scale is in units of kB T.

As shown in Fig. 15, hydrophobic CG-SASA overall decreased with oligomer order for Xlinked and non-X-linked oligomers of both alloforms. The effect of X-linking on hydrophobic CG-SASA can be quantified by comparing one-dimensional hydrophobic CG-SASA distri38


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butions of X-linked and non-X-linked oligomers (Fig. S7). Fig. S7 reveals that the effect of broadening of the hydrophobic CG-SASA range due to X-linking was stronger in Aβ42 than in Aβ40 . The one-dimensional distribution of hydrophobic CG-SASA values showed multiple peaks except Aβ40 hexamers. Mann-Whitney U tests comparing non-X-linked with X-linked distributions showed statistically significant difference for all oligomer sizes. Xlinking broadened the range of hydrophobic CG-SASA values for Aβ40 dimers and trimers and for Aβ42 dimers, trimers, and hexamers (Fig. S7), indicating a more diverse population of conformations with respect to solvent accessibility of hydrophobic residues. X-linking also increased the average hydrophobic CG-SASA values in Aβ40 and Aβ42 dimers, trimers, and hexamers. The average hydrophobic CG-SASA decreased with oligomer order for both X-linked and non-X-linked oligomers of the two alloforms. As in the case of non-X-linked oligomers, X-linked Aβ42 oligomers had on average larger hydrophobic CG-SASA values than X-linked Aβ40 oligomers (Table S1). A similar broadening and splitting of the conformational space due to X-linking was noted for the N-CM distance distributions in Aβ40 and Aβ42 dimers, trimers, and hexamers. Fig. S8 displays one-dimensional distributions of the N-CM distances corresponding to data shown in Fig. 15. These distributions exhibit multiple peaks corresponding to multiple free energy minima in Fig. 15, which indicate co-existence of multiple distinct locally stable conformations. Overall, X-linking increased polymorphic nature of oligomers of both alloforms, but this effect was particularly strong in Aβ42 oligomers. The comparison of the X-linked and non-X-linked distributions of the N-CM distance by Mann-Whitney U test again showed that X-linking affected these distributions in a statistically significant way. The average NCM distance also increased for all Aβ40 and Aβ42 oligomers (Table S1). This difference was larger in trimers and hexamers than in dimers of both alloforms. We also examined the one-dimensional distributions of the C-CM distance. One-dimensional distributions of the C-CM distance for X-linked and non-X-linked Aβ40 and Aβ42 dimers, trimers, and hexamers are shown in Fig. S9. Mann-Whitney U-test was applied to ex-

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amine whether the differences in the C-CM distance distributions between X-linked and non-X-linked oligomers were statistically significant, and the results revealed that X-linking significantly altered these distributions as well. However, in contrast to the average N-CM distance, which strongly increased due to X-linking, the average C-CM distance was not strongly affected. Non-X-linked and X-linked Aβ40 and Aβ42 oligomers were characterized by overall comparable average C-CM distances. The representative conformations of X-linked and non-X-linked Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers corresponding to the distinct PMF minima are displayed in Fig. 15. Unlike simple X-linking via tyrosines which resulted in dumbbell-like shaped Xlinked Aβ dimers and trimers with tyrosines near the CM, 37 oligomers X-linked via mechanism 3 were visually more similar to non-X-linked oligomers. The X-linked oligomers were observed to form globular shapes that resembled non-X-linked oligomer conformations as well as more elongated shapes with spread NTRs, consistent with the increased N-CM distance and the CG-SASA at the NTR due to X-linking. The NTRs of neighboring peptides in the oligomer were prone to form parallel strand structures, especially in Aβ42 hexamers.

Conclusions Aβ40 and Aβ42 oligomers are hypothesized to trigger the cascade of events that leads to neurodegeneration in AD yet their structure remains elusive. PICUP is an important X-linking technique that induces covalent bonds among proximate peptides in oligomers, thereby allowing the quantitative in vitro characterization of the respective oligomer size distributions using SDS-PAGE. 10,11 Whereas PICUP chemistry is not physiologically relevant, another X-linking technique, CHICUP, which produces similar Aβ40 and Aβ42 oligomer size distributions as PICUP, is consistent with the chemistry of the AD brain. 20 Either of the two X-linking techniques, PICUP or CHICUP, stabilizes Aβ40 and Aβ42 oligomers, which prolongs their ability to disrupt membranes. 20 Although it is not known whether stabilization

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of Aβ oligomers via CHICUP occurs in the AD brain, there is evidence that Aβ can form dityrosine bonds in vivo. Al-Hilaly et al. showed that dityrosines co-localize with intracellular Aβ in lysosomes and have been detected also in extracellular amyloid plaques of AD patients. 50 It has been thus proposed that X-linked Aβ40 and Aβ42 oligomers may be the proximate neurotoxic species in AD. 23 DMD4B-HYDRA simulations of Aβ40 and Aβ42 oligomer formation have been shown to capture alloform-specific oligomer size distributions and distinct structural features Aβ40 and Aβ42 oligomers. 25,30 Recently, the effect of simple X-linking via tyrosines on Aβ40 and Aβ42 oligomer formation and structure was explored using the DMD4B-HYDRA approach. 37 The resulting Aβ40 and Aβ42 oligomer size distributions turned out to be unstable and no X-linked oligomers larger than Aβ40 trimers and Aβ42 tetramers were observed, 37 which is inconsistent with experimental findings showing that Aβ40 and Aβ42 form X-linked oligomers larger than trimers and tetramers, respectively, and that oligomers in X-linked samples remain relatively small and quasi-spherical as observed by AFM. 20 The above simulation results thus strongly suggest that amino acids other than tyrosine are involved in X-linking. Here, we examined three distinct X-linking mechanisms involving in addition to formation of YY covalent bonds also formation of YK covalent bonds (mechanism 1), HH covalent bonds (mechanism 2), and conversion of F into hydroxylated F’, followed by formation of YF’ and F’F’ covalent bonds (mechanism 3). Simulations were performed in two stages to mimic in vitro preparation of the Aβ sample in a buffer (stage 1) followed by X-linking (stage 2), as described previously. 37 Whereas X-linking simulations in stage 2 were rather straightforward in mechanisms 1 and 2, mechanism 3 involved a two-step process (stages 2-1 and 2-2). In stage 2-1, F was converted into non-hydrophobic F’ and only interpeptide YY covalent bonds were enabled, whereas in stage 2-2, all Y and F’ amino acids were involved in intraand interpeptide X-linking. The three X-linking mechanisms were assessed by monitoring two types of oligomer size distributions during X-linking (stage 2 of simulations) through a comparison with the respective quantities derived from the corresponding non-X-linked

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(control) simulations. The ”regular” oligomer size distribution did not distinguish between covalent and non-covalent bonds, whereas the X-linked oligomer size distribution was based on covalent bonds only to mimic the SDS-PAGE process. Our results revealed that both Xlinking mechanisms 1 and 2 resulted in unstable oligomer size distributions whereby X-linking induced formation of large oligomers, inconsistent with experimental findings. In addition, the X-linked oligomer size distribution of Aβ42 did not display a multimodal character under mechanism 1 or 2, which also disagrees with in vitro measurements. In contrast, X-linking via mechanism 3 produced stable Aβ40 and Aβ42 oligomer size distributions and also captured the multimodal character of X-linked Aβ42 oligomer size distribution. Our results clearly show that X-linking via tyrosines alone in stage 2-1 produces mostly X-linked dimers and trimers and that stage 2-2 is essential for formation of larger X-linked oligomers, such as e.g. Aβ40 tetramers and Aβ42 pentamers and hexamers. We thus conclude that of the three mechanisms under investigation, only mechanism 3 is consistent with experimental observations. We then characterized Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers X-linked via mechanism 3. Structural analysis revealed that X-linking via mechanism 3 increased turn and coil content, and significantly decreased β-strand content in X-linked oligomers. X-linking inhibited both tertiary and quaternary contacts involving the NTR and F’19-F’20 region due to non-hydrophobic character of F’ (in contrast to hydrophobic F). Interestingly, these changes resulted in stronger interpeptide contacts among the MHR and CTR in oligomers of both alloforms. Consequently, the solvent exposure in the NTR increased in X-linked oligomers relative to their non-X-linked counterparts. X-linking pushed the NTR farther away from the CM, broadened the free energy landscapes of Aβ40 and Aβ42 conformations relative to the corresponding non-X-linked conformations, and thereby increased polymorphic nature of Aβ40 and even more so Aβ42 oligomer conformations. Specifically, Aβ42 exhibited pairs or even triplets of NTRs sticking out of quasi-spherical to elongated conformations.

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Why did only mechanism 3 produce stable Aβ40 and Aβ42 oligomer size distributions and lead to X-linked oligomer size distributions consistent with experimental findings? One difference between mechanism 3 and the other two mechanisms is that F’19 and F’20 involved in X-linking are within the CHC, which was already close to the CM in non-X-linked oligomers. During stage 1, when Aβ peptides self-assembled in the absence of X-linking, the CHC was one of the regions that were closest to the CM. During stage 2, amino acids involved in X-linking were pulled closer to each other when forming covalent bonds, thereby driving other amino acids farther away from the CM. Although in Aβ40 and Aβ42 oligomers X-linked via mechanism 3, due to the lack of hydrophobic character of F’, the CHC became more exposed to the solvent and farther from the CM, the overall arrangement of amino acids was not strongly altered relative to matching non-X-linked conformations, which contributed to stabilization of oligomer size distributions as well as emergence of X-linked oligomers larger than those produced by simple X-linking via tyrosines. Mechanism 3 is also consistent with qualitative observation that upon substituting Y by F in Aβ40 and Aβ42 , the X-linking efficiency was reduced by 30-50% but did not go to zero. 49 It will be important to perform in vitro experiments to test whether or not X-linking indeed involves hydroxylation of phenylalanine into tyrosine, which may be possible to determine using mass spectrometry. X-linking via mechanism 3 induced changes in the secondary, tertiary, and quaternary structure of Aβ oligomers. The increased solvent exposure at the NTR, which was more pronounced in larger oligomers, such as Aβ42 hexamers, is particularly noteworthy. Increased flexibility and solvent exposure at the NTR, which was first observed in DMD4B-HYDRA simulations more than a decade ago, 25 was proposed to underlie increased ability of Aβ42 over Aβ40 oligomers to mediate toxicity, possibly through interactions with a cellular membrane. 23 Additional DMD4B-HYDRA studies of Aβ isoforms relevant to AD 30,31,51 and Aβ42 oligomer formation in the presence of effective and ineffective toxicity inhibitors 52 provided even more evidence in favor of this potential structure-toxicity relationship. A recent study of fully atomistic Aβ40 and Aβ42 oligomers demonstrated that this feature at the NTR is intimately

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linked to the ability of the oligomer to form water permeable pores. 36 Importantly, the propensity for pore formation sharply increased with oligomer order. 36 If embedded into a membrane, such pores could act as ion channels, resulting in an abnormal calcium flux and thereby causing cell death. Our present results thus suggest that X-linking may enhance the ability of oligomers to form pores and, by implication, ion channels, which is consistent with the structure-toxicity conjecture that links flexibility and solvent exposure of NTR to the ability to mediate toxicity. These findings provide a plausible explanation of why the toxicity of X-linked Aβ40 oligomers in cell cultures sharply increases with oligomer order as reported by Ono, Condron, and Teplow. 53 Overall, this study combined with previous findings suggests that small molecules that bind the NTR of X-linked Aβ oligomers may be efficient inhibitors of Aβ oligomer-induced toxicity.

Associated Content As supplementary of the results displayed above, we showed the average and SEM values of hydrophobic CG-SASA, N-CM distance, and C-CM distance of non-X-linked and Xlinked Aβ40 and Aβ42 monomers, dimers, trimers, and hexamers in a table. And we plotted comparison between Aβ40 and Aβ42 X-linked oligomer size distributions, the turn, β-strand, and coil propensities per residue, amino acid-specific distance to the CM in YY X-linking mechanism, probability distribution of hydrophobic CG-SASA, N-CM distance, and C-CM distance of X-linked and non-X-linked monomers, dimers, trimers, and hexamers of Aβ40 and Aβ42 .

Acknowledgment We thank Prof. Monica Ilies (Department of Chemistry, Drexel University) for her valuable insights into the chemistry of covalent bond formation under oxidative stress conditions. This work was not supported by any external funding. 44


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Elucidating the Role of Hydroxylated Phenylalanine in the Formation

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