TYPE Original Research PUBLISHED 06 February 2023 DOI 10.3389/fnins.2023.1110311 Computational assessment of the OPEN ACCESS impact of Cu(II) and Al(III) on β EDITED BY Jinghui Luo, Paul Scherrer Institut (PSI), Switzerland -amyloid42 fibrils: Binding sites, REVIEWED BY Jorge Alí-Torres, structural stability, and possible National University of Colombia, Colombia Jamie Platts, Cardiff University, United Kingdom physiological implications *CORRESPONDENCE Mariona Sodupe Lorena Roldán-Martín, Mariona Sodupe* and mariona.sodupe@uab.cat Jean-Didier Maréchal Jean-Didier Maréchal* jeandidier.marechal@uab.cat Departament de Química, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain SPECIALTY SECTION This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience One of Alzheimer’s disease major hallmarks is the aggregation of β-amyloid peptide, RECEIVED 28November 2022 a process in which metal ions play an important role. In the present work, an ACCEPTED 10 January 2023 PUBLISHED 06 February 2023 integrative computational study has been performed to identify the metal-binding CITATION regions and determine the conformational impact of Cu(II) and Al(III) ion binding to Roldán-Martín L, Sodupe M and Maréchal J-D the β-amyloid (Aβ42 ) fibrillary structure. Through classical and Gaussian accelerated (2023) Computational assessment of the impact of Cu(II) and Al(III) on β -amyloid42 molecular dynamics, it has been observed that the metal-free fiber shows a hinge fibrils: Binding sites, structural stability, fan-like motion of the S-shaped structure, maintaining the general conformation. and possible physiological implications. Upon metal coordination, distinctive patterns are observed depending on the metal. Front. Neurosci. 17:1110311. doi: 10.3389/fnins.2023.1110311 Cu(II) binds to the flexible N-terminal region and induces structural changes that COPYRIGHT could ultimately disrupt the fibrillary structure. In contrast, Al(III) binding takes place © 2023 Roldán-Martín, Sodupe and Maréchal. with the residues Glu22 and Asp23, and its binding reinforces the core stability of the This is an open-access article distributed under the terms of the Creative Commons Attribution system. These results give clues on the molecular impact of the interaction of metal License (CC BY). The use, distribution or ions with the aggregates and sustain their non-innocent roles in the evolution of the reproduction in other forums is permitted, provided the original author(s) and the illness. copyright owner(s) are credited and that the original publication in this journal is cited, in KEYWORDS accordance with accepted academic practice. No use, distribution or reproduction is molecular modeling and simulation, molecular dynamic (MD), amyloid Aβ-42, protein-ligand permitted which does not comply with docking, metal these terms. Introduction Nowadays, neurodegenerative diseases are hot topics in a broad spectrum of the scientific community. The most predominant one is Alzheimer’s disease (AD) with six out of 10 dementia cases (WHO, 2019). AD first affects the memory areas but as it spreads, other areas of the brain get damaged including those controlling language, reasoning, and cognitive skills. Many efforts have been placed into discovering the onset of the disease and how to reverse it, but no effective treatments have been proposed so far (Breijyeh et al., 2020). Amyloid plaques and neurofibrillary tangles are the main hallmarks of AD and are caused by the accumulation of β-amyloid (Aβ) and tau protein, respectively (Kepp, 2012; Deture and Dickson, 2019). How these proteins start their aggregation process is still widely unknown. Aβ arises from the cleavage of the amyloid precursor protein (APP) and several hypotheses exist regarding its aggregation mechanism. The most accepted is the amyloid cascade (Hardy and Higgins, 1992), which suggests that the aggregation of Aβ, arising from an imbalance between Aβ production and clearance, is the main causative agent of Alzheimer’s disease Frontiers in Neuroscience 01 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 pathology. Nonetheless, such aggregation is also observed in healthy Materials and methods patients (Bennett et al., 2006). Another possibility is the metal ion hypothesis (Maynard et al., 2005), derived from the high The computational protocol followed in this study is summarized concentration of metal ions [Zn(II), Fe(III), Al(III), and Cu(II)] in in Figure 1. Aβ plaques, which postulates that a loss of metal homeostasis could enhance the aggregation process. Regardless of the onset process, once the aggregation has started, the monomeric forms expand Initial models forming oligomers, the most neurotoxic forms, that bind together to form protofibrils and ultimately the entire fibrils. Along all these Several fibrillary structures of the β-amyloid fibril are available stages, though, the aggregation unit can be broken producing smaller in the Protein Data Bank. The most complete PDB structure, with subunits with seed properties capable of inducing disease in new code 2NAO (Wälti et al., 2016), corresponds to the sequence 1–42 of the amyloid for each strand, though it retrieves poor punctuation areas. Accordingly, it is extremely important to properly understand in all the PDB parameters (clashes score 22, Ramachandran 12.4% the different phases of the aggregation process to produce effective and Sidechain 26.7%). On the other hand, the PDB structure with drugs either to prevent the raise of the disease or slow down its code 2MXU (Xiao et al., 2015) presents far better punctuation advance (Kepp, 2012). (clashes score 0, Ramachandran 3.8% and sidechain 6.22%) at In the last decades, numerous studies have focused on decoding the expense of residues 1 to 11 being missing, an observation the folding and aggregation process of Aβ systems (Groh et al., consistent with the high flexibility of the NTer region of the peptide. 2017; Almeida and Brito, 2020; Ke et al., 2020; Krishnamurthy et al., Therefore, for this study, we selected the latest and elongated each 2022; Rahman et al., 2022). Despite these efforts, the elucidation strand with the 11 missing residues using homology modeling at the molecular level of the aggregation mechanism has not been through the UCSF Chimera Software Modeler tool (Pettersen reached yet. For monomers, the major challenge arises from their et al., 2004) to obtain the N-terminal missing region. Chimera intrinsic flexibility, becoming even more complex when they are minimization was performed to reduce the clashes, with the Amber merged with metal ion binding. To date, several experimental (Drew ff14SB force field (Maier et al., 2015) and 100 steps of steepest and Barnham, 2011; Faller et al., 2013; Stefaniak et al., 2021) and descent. theoretical (Huy et al., 2016; Liao et al., 2018; Mutter et al., 2018; Once the complete fibrillary structure was obtained, several Pham et al., 2018; Khatua et al., 2019; Strodel and Coskuner-Weber, preliminary molecular dynamics calculations were performed. We 2019; Turner et al., 2019; Boopathi et al., 2020; Roldán-Martín et al., considered the systems with different numbers of strands, namely 4, 2021) studies have been performed with respect to different metals 6, 8, and 10 aiming at identifying the minimal number of strands (Cu, Zn, and Al) binding to monomeric Aβ. In our previous study, necessary for the fiber to remain stable in the fibrillar form during the trajectory. This is also particularly important to avoid possible it was demonstrated that monomeric Aβ can adopt a U-shaped drawbacks inherent to force field propensity to stabilize certain structure with two antiparallel α-helix regions, whose secondary secondary structures against others (Case et al., 2005). With the and tertiary structure can be modified upon Cu(II) and Al(III) Amber force field, we observed that at least 6 strands are necessary coordination, increasing β-sheet content in the latter case (Roldán- to obtain a fibril, though at least 10 strands should be considered for Martín et al., 2021). a stable core. In this study, only the fibrillary structure of 10 strands However, when it comes to larger aggregated forms such as is discussed. fibrillary Aβ complexes, the experiments lack the techniques to precisely identify the binding site of metal ions (Miller, 2022). Therefore, there are scarce experimental details on plausible metal- Metal-binding areas: BioMetAll and GOLD binding sites in fibrils (Parthasarathy et al., 2011), and computational approaches have been shown as suitable techniques to determine Putative metal-binding areas were detected by applying the the impact of metal binding on Aβ fibril dynamics. Contrary to BioMetAll software (Sánchez-Aparicio et al., 2021) to the Aβ11−42 monomers, aggregate forms are less flexible and several forms of crystallographic structure available in the PDB 2MXU, which was fibrillary structures have been recently reported by X-ray and ss- configured to include the backbone atoms and a minimum of NMR techniques (Xiao et al., 2015; Wälti et al., 2016; Kollmer et al., three residues as possible coordinating groups. BioMetAll only 2019; Liberta et al., 2019), providing a solid ground for computational considers the backbone and the residues that are within a suitable studies to unravel the effect of metal ion binding to amyloid fibrils. distance to coordinate the metal, but their orientation may not be optimized for coordination. Therefore, protein-ligand dockings To shed light on those questions, we decided to apply a multi- calculations were performed with the program GOLD (Jones et al., scale protocol focusing on Cu(II) and Al(III) bound forms of Aβ42 1997) using the GoldScore scoring (Jones et al., 1995) function, fibril, which are known to be more aggregation-prone. The work whose parameter file was modified to include atom types for metal stands on (1) BioMetAll (Sánchez-Aparicio et al., 2021), a recently ions and their possible coordinating amino acids (Sciortino et al., released structural predictor of metal-binding sites in proteins, (2) 2018). Genetic algorithm (GA) parameters were set to 50 GA protein-ligand dockings compatible with metallic ligands (Sciortino runs and a minimum of 100,000 operations each. The remaining et al., 2018), and (3) classical and accelerated molecular dynamics. parameters, including pressure, number of islands, crossovers, or The impact of the metal on Aβ fibril formation and stabilization mutations, were set to default. Finally, docking solutions were is assessed with the purpose of providing structural knowledge that analyzed through GaudiView (Rodríguez-Guerra Pedregal et al., could help in understanding the role of metal in the aggregation 2017) an in-house developed GUI tool built as an extension of processes associated with Alzheimer’s disease. UCSF Chimera. Frontiers in Neuroscience 02 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 FIGURE 1 Methodological protocol used in the present study. Classical molecular dynamics simulations (Rassolov et al., 2001). For Cu(II) complexes, we used the for metalloaggregates 6-31 + G (d,p) basis set for C, H, N, and O atoms; and the SDD pseudo-potential and the corresponding basis-set supplemented Metal parameters were obtained using the MCPB.py package with f-polarization function for Cu (Ehlers et al., 1993). Solvent- (Li and Merz, 2016) from quantum mechanical calculations with polarizable dielectric continuum model (SMD) was considered to DFT (B3LYP) (Lee et al., 1988; Becke, 1998) and adding Grimme’s account for the solvent effects in water (Marenich et al., 2009). Force D3 correction for dispersion (Grimme et al., 2010). For aluminum constants and equilibrium parameters for metal-coordinating atoms complexes, the 6-31 + G (d,p) basis set was used for all atoms were obtained through the Seminarios’s method (Seminario, 1996) Frontiers in Neuroscience 03 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 while point charges were derived using the RESP (restrained first present the study of the metal-free fiber to generate a point of electrostatic potential) model (Bayly et al., 1993). comparison and then pursue metal-bound systems. Molecular dynamics simulations were then performed with AMBER18 (Case et al., 2018) using the AMBER ff14SB (Maier et al., 2015) force field in the NPT ensemble, with a 1 fs integration Dynamical behavior of metal-free fibril time step. The initial models were embedded within a cubic box of pre-equilibrated TIP3P (Jorgensen et al., 1998) water molecules The initial structure of the metal-free fibril has been obtained and Na+ ions were included to balance the total charge depending from the solid-state NMR (ss-NMR) structure of Aβ11−42 (pdb code on the system: 30 for the metal-free system, 20 for the copper- 2MXU). Since the NTer end of the system is missing (residues 1 to 10), bound system, and 15 for the aluminum-bound system. Constant the structure was completed by homology modeling using modeller temperature and pressure were set by coupling the system to a Monte (Martí-Renom et al., 2000; Figure 2A). Then, long GaMD simulations Carlo barostat (Duane et al., 1987) at 1.01325 bar and a Langevin were performed using a biased potential and the results were analyzed thermostat at 300 K (Loncharich et al., 1992). The SHAKE algorithm using several statistical tools. (Ryckaert et al., 1977) was used to constrain the bonds involving The total 3 µs GaMD simulation spread over three replicas hydrogen atoms. Classical MD simulations were performed for 10 of 1 µs. The three replicas exhibit similar behaviors in terms ns for all the systems, just to equilibrate the system before running of global breathing motions and exhaustive exploration of the Gaussian accelerated molecular dynamics (GaMDs). Moreover, MD conformational space. Accordingly, PCA and energetic profiles simulations have also been performed on the lowest energy wells of the replica with the lowest energetic conformation identified obtained in the GaMD simulations. are presented in the main text (Figures 2B, C) while the other ones are reported in ESI (Supplementary Figure 1). Principal component analysis shows a displacement of the system at the GaMDs simulations end of the first 100 ns, then, it explores a limited region of the space until approximately 600 ns, and finally, it explores Enhanced conformational sampling was performed with the back the previous region, so that the simulation is converged Gaussian accelerated molecular dynamics (GaMDs) (Miao et al., (Figure 2B). The potential energy profile clearly indicates that during 2015). For these simulations, the AMBER ff14SB force field in the the trajectory, a series of low-energy wells are explored with a NVT ensemble was used, constraining the bonds involving H atoms radius of gyration slowly raising–meaning a geometric expansion with SHAKE, with an integration time step of 2 fs. A boost on both of the system (Figure 2C). This is in line with the results reported dihedral and total potential energy (igamd = 3) was applied. In total, by Miller et al. (2012), who suggest that fibers do not show a three GaMD replicas of 1 µs were produced for each system. unique lower energy state but different conformers with barriers between them, which are expected to be more or less favorable upon metal binding. Analysis The trajectories present a series of interesting features. The first one is that only the six central strands maintain the fibrillary The energy of the systems was calculated along the trajectory with structure while the two at each extreme tend to unfold. This is related the Generalized Born Implicit model (Onufriev et al., 2000, 2004), for to the fact that these extremes are more exposed to the solvent 1,000 frames extracted from each 1 µs GaMD trajectory, stripping and naturally start to adopt a more molten globule geometry with water molecules, and performing a short minimization (maxcyc = 500 some helical contents, a situation reminiscent of the simulations steps) previously to the energy extraction. The radius of gyration of monomeric peptides. The second is that the terminal regions of (RoG) was computed over the same 1,000 frames, so a graphic with each strand represented by the amino acids from 1 to 10 are overall the different minima was obtained. For each system, the well with the disorganized. This is consistent with the absence of atomic density minimum energy was selected for an MD, to test its stability, and for in the pdb structure and suggests that the overall geometry of the the analysis. amyloid is barely affected by the NTer region adopting quite random Principal component analysis (PCA) (Wold et al., 1987), RMSD conformations. Based on this, the NTer region and the four strands all-to-all, and clustering were performed over both the GaMD and at the extremes are discarded for the rest of the analyses, which MD simulations to test whether the exploration was exhaustive focuses on the core formed by the six central strands and from enough. For the MD simulations performed on the lowest energy residues 11 to 42. wells from the GaMDs, the RMSD with respect to the initial structure The major motion observed is a fan-like movement of the was also computed. The last analysis performed consisted of studying Aβ11−20 β-sheet, which appears to be the natural breathing the PCA movements of the fibers along the trajectories with the movement of the system (Figure 2D) as counterchecked by normal VMD tool NMWiz and counterchecked by normal mode analysis mode analysis performed using WEBnma software (Tiwari et al., performed with WEBnma software (Humphrey et al., 1996; Bakan 2014). This fan-like motion consists of a hinge that can be defined as et al., 2011) to study the global low-energy internal motions of the the angle between the principal axes of the sets of atoms of residues 11 systems. to 15 and 39 to 41 (Figure 2E). The angle obtained between these two principal axes for a representative structure of the most populated cluster reaches 48.5◦ against the 7.8◦ of the ss-NMR structure. For Results and discussion the lowest energy structure, the same analysis shows a hinge of 46.7◦ (Figure 2F), which still represents the main distortion with respect to This work aims at investigating the effect of the binding of Cu(II) the ss-NMR structure. These simulations and analyses show that the and Al(III) to the Aβ1−42 fibers through molecular modeling. We metal-free fiber mostly retains the experimental geometry although it Frontiers in Neuroscience 04 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 FIGURE 2 (A) Aβ42 model built from Aβ11−42 (2MXU) (Xiao et al., 2015) after adding the 1–11 residues, (B) principle component analysis (PCA) of Gaussian accelerated molecular dynamics (GaMD) simulation, (C) energy profile and radius of gyration (RoG) along the GaMD simulation, (D) overlap of representative structures of most populated clusters obtained from GaMD simulations, (E) in blue, metal-free fibril axis representation of 39–41 (top) and 11–15 β-sheets (bottom), in green; 11–15 β-sheets axis of NMR-structure, and (F) overlap between the solid-state NMR (ss-NMR) structure and the lowest energy structure of the GaMD simulation. can display the dynamic motions not observed in the experimental the search was first performed looking for a minimum of three structures. coordinating amino acids and any amino acid known to participate in the coordination sphere of transition metals. With this screening, BioMetAll finds the geometries compatible with the simultaneous Predicting metal-binding sites in amyloid coordination of two His residues in the NT er region of the Aβ fibril aggregated structures as identified in the monomers (Alí-Torres et al., 2011, 2014) from the experiments and QM calculations. However, this only occurs in a The binding of metal ions to amyloids has been the source of few strands due to the intrinsic flexibility of that region. Since other an extensive number of studies (Faller, 2009; Mutter et al., 2018; metal-binding sites are more frequently found in the core region Pham et al., 2018; Roldán-Martín et al., 2021; Miller, 2022). Today, of the fibril, indicating that in this system, the situation is different a relatively clear picture of metal binding has emerged in the from what happens in monomers, we discarded NT er monomer- monomeric systems, either theoretically for Al(III) (Mujika et al., like binding sites and focused on the core region, favoring Asp and 2017) or experimentally for Cu(II) (Drew and Barnham, 2011), which Glu for Al(III) (Mujika et al., 2017) and His and Gln for Cu(II). has narrowed the location and type of amino acids that bind the For the latter, it is important to notice that Gln residues have been metal ions with only few candidates, all part of the NTer region of included in the search since, although rare, the coordination of Gln the peptide. For aggregates, though, there is still a substantial level of to Cu(II) is observed in a series of X-ray structures from the protein uncertainty. data bank, it has also been proposed as a possible coordinate residue The previous part of our study sustains an intrinsically disordered (Parthasarathy et al., 2011), and Gln15 shows the proper location for protein (IDP) behavior of the NTer region of the aggregate. This metal coordination in the selected structures. flexibility indicates that the binding of the metal ions to this region In total, two areas were found for metal binding in the core of would be similar to what occurs in monomeric species. It also the fibril: one consistent with copper and the other with aluminum. shows that the core of the aggregate is quite rigid, and one would The former, named CuF , stands at the linker between the NTer loop naturally wonder if this rigidity induces some pre-organizations and the β-strand regions (from residue 12–15) and is composed of for metal binding. histidine His13 of two adjacent strands and two glutamine Gln15 of To identify putative metal-binding sites in the aggregates, we the same strands (Figure 3A). The latter, named AlF , is located at performed BioMetAll calculations, a software recently released by the beginning of the coil with residues Glu22 and Asp23 of adjacent our group for the prediction of metal-binding sites in biomolecules. strands (Figure 3B). This latter site agrees with the work reported by Based on the backbone pre-organization hypothesis, BioMetAll has Yang et al. (2022) which shows that this area is likely to be bound to shown excellent success rates in identifying metal-binding locations a metal ion. With these two sites identified, we pursued generating for a wide sample of proteins and ions (Sánchez-Aparicio et al., 2021). realistic three-dimensional models of the metal-aggregate systems by To search for site-specific binding areas for copper and aluminum, carrying out protein-ligand dockings. Those were performed with Frontiers in Neuroscience 05 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 FIGURE 3 BioMetAll results of Cu(II) (A) and Al(III) (B) coordination. Once the areas were identified, docking results with Gold software were obtained for Cu(II) (C) and Al(III) (D). GOLD, assuming a square planar environment for Cu(II) and an metal-free fiber appears to be reduced, the angle between the two octahedral one for Al(III) (Figures 3C, D). principal axes (considering the six central strands as done previously The results indicate that Cu(II) can achieve a square planar for the metal-free fiber) in a representative structure of the most coordination with two His and two Gln from adjacent strands. populated cluster being 6.1◦ (Figure 4E). Note that in this case, the However, for Al(III), docking results only find an incomplete motion is not globally spread all other the fibril since there is a octahedral coordination in which two Glu and two Asp of adjacent disruption of the fibrillar form that arises from a higher displacement strands are involved, thereby leaving vacant sites that would probably of the first strands with respect to the remaining ones, probably due be occupied by the solvent molecules. Each model was then submitted to the repulsion between Cu(II) ions (Figure 4F). Such observations to triplicated Gaussian accelerated molecular dynamic simulations to may indicate that Cu(II) binding tends to disrupt the fibrillar form evaluate its stability and dynamics properties as well as to compare to lower-aggregation structures or amorphous aggregates without with the metal-free system. fibrillary structure. For Al(III), the preferred binding site in the fiber involves the residues Glu22 and Asp23 of adjacent strands (Figure 5A). In this GaMD of metal bound fibers last case, the GaMD explores a series of minima until finding a well it cannot escape during the last 400 ns (Figure 5B and Supplementary Data of the triplicated simulations on CuF are given in Figure 4 Figure 3). Despite the energy profile highlighting a remarkable and Supplementary Figure 2 of ESI. In all cases, the trajectories minimum (Figure 5C), the whole trajectory displays a very stable tend to converge around 400 ns (Figure 4B and Supplementary fold with minor motions (Figure 5D). In particular, the amplitude Figures 4D–F) and show the presence of a series of minima of the fan-like movement is far more limited in AlF than in the (Figure 4C). Such minima agree with the fact that amyloids are other two systems. For example, one of the most representative highly polymorphic, metal binding shifting the population toward a structures presents an inter-axial angle of only 2.7◦ , similar–even certain conformation (Miller et al., 2010). Overall, the 3 µs GaMD lower–than that in the ss-NMR structure (7.8◦ ) (Figures 5E, F). simulations depict the same fan-like motion observed in the metal- Therefore, GaMD simulations show that the aluminum-bound free fiber (Figure 4D). Strikingly though, the geometry variability of aggregate displays a more compact structure than both free-metal and the NTer loop (residues 1–11) is significantly more pronounced in copper-bound ones and remains closer to the static view provided by CuF ; i.e., each metal-bounded pairs of strands have their motions experimental ss-NMR geometry (Figure 5F). uncoupled from adjacent pairs. Consequently, the β-sheet H-bonds between these adjacent pairs are significantly weakened, which favors the change in their secondary structure increasing the α-helix content Further analysis in the NTer ends, particularly on the first strands. Such α-helix regions are reminiscent of the monomeric forms, a behavior more To further analyze how the metal ions impact the conformation pronounced than in the metal-free system (Roldán-Martín et al., landscape and stability of the aggregates, classical MDs of 100 ns 2021). However, the hinge movement previously observed in the were carried out starting from the structure of the lowest energy well Frontiers in Neuroscience 06 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 FIGURE 4 (A) Aβ42 -Cu(II) model, (B) principle component analysis (PCA) of Gaussian accelerated molecular dynamics (GaMD) simulation, (C) energy profile and radius of gyration (RoG) along the GaMD simulation, (D) overlap of representative structures of most populated clusters obtained from GaMD simulations, (E) in blue, axis representation of 39–41 residues (top) and 13–16 β-sheets (bottom); in green; 13–16 β-sheets axis of NMR-structure, and (F) overlap between the solid-state NMR (ss-NMR) structure and the lowest energy structure of the GaMD simulation. FIGURE 5 (A) Aβ42 -Al(III) model, (B) principle component analysis (PCA) of Gaussian accelerated molecular dynamics (GaMD) simulation, (C) energy profile and radius of gyration (RoG) along the GaMD simulation, (D) overlap of representative structures of most populated clusters obtained from GaMD simulations, (E) in blue, axis representation of 39–41 residues (top) and 13–15 residues β-sheets (bottom); in green; 13–16 β-sheets axis of NMR-structure, and (F) overlap between the solid-state NMR (ss-NMR) structure and the lowest energy structure of the GaMD simulation. obtained from the GaMD simulations. To test the exhaustiveness of breathing motions. For that, five distances were evaluated using the exploration and their stability, RMSD and PCA were performed the carbon α of each residue and considering only the six central on the trajectories (Supplementary Figure 4). Emphasis is given strands of the fibril. To ease the labeling and the reading, we use the to how metal binding alters the fiber’s low collective modes and X-ResNum identification format where X is the number of the strand Frontiers in Neuroscience 07 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 FIGURE 6 Atoms chosen for the horizontal distance (A), vertical distance (B), 11–15 β-strand intersheet distance (C) and opening of the 1–18 region (D) represented in the solid-state NMR (ss-NMR) model. Results of the horizontal (E), with an average distance of 24.6 (± 5.6)Å, 25.2 (±1.6)Å and 25.5 (±10.9)Å for metal-free, Cu(II) and Al(III) systems; vertical (F), with an average distance of 19.9 (±0.3)Å, 18.7 (±0.2)Å and 18.8 (±0.2)Å for metal-free, Cu(II) and Al(III) systems; intersheet (G); with an average distance of 19.3 (±5.2)Å, 26.06 (±1.1)Å and 21.5 (±0.7)Å for metal-free, Cu(II) and Al(III) systems; and opening (H), with an average distance of 32.1 (±0.4)Å, 29.6 (±0.5)Å and 23.1 (±0.4)Å for metal-free, Cu(II) and Al(III) systems; along the MD simulation, with ss-NMR distance represented in yellow line. the residue belongs to, Res is the type of the residue, and Num is its Measure 3 relates to the disruption of the fibrillary structure in number in the sequence. the NTer region. It is observed that Cu(II) coordination produces an increase in this distance, from 24.3 Å in the ss-NMR to 26 (±1.1) Å. In contrast, both the metal-free fibril and Al(III)-bound structure show 1. The distance between 3-Gly33 and 8-Gly33 measures how the shorter distances, of 19.93 (±0.3) Å and 21.5 (±0.7) Å, respectively, fiber opens horizontally in the core region and indicates if the β- in comparison with the ss-NMR structure (Figure 6G). Measure 4, on sheet interaction is weakened (longer distance) or strengthened its side, is remarkably increased in the metal-free fibril [32.1 (± 0.4) (shorter distance) (Figure 6A). Å] with respect to the ss-NMR (29.3 Å), due to the hinge fan-like 2. The average distance from Phe19 to Val39 of each core movement. Cu(II) system is maintained in a similar value to the ss- strand indicates how the fiber opens vertically and measures NMR structure, with a distance of 29.6 (±0.5) Å. Note, however, the compactness of the S-shaped supramolecular structure that this distance is an average of six values and the displacement (Figure 6B). of the terminal strands is faded by the shorter distance retrieved 3. The distance between 3-Gln15 and 8-Gln15 measures how the by the other ones. Indeed, the two most external strands included fiber opens horizontally in the NTer region and is indicative of in the average exhibit values of 35 and 42 Å, respectively, whereas the β-sheet interaction at this region (Figure 6C). the four inner ones show values that range between 20 and 25 Å. 4. The average distance from Glu11 to Val39 of each core strand Finally, the Al(III) system is even more compact than the ss-NMR describes the separation movement of the NTer tail from the core structure, with an average distance of 23.1 (±0.4) Å, supporting the region of the fiber (Figure 6D). fact that aluminum binding increases the stability of the fibrillary 5. The average distance between His14 of each core strand conformation (Figure 6H). describes if the fiber movement is collective (equal distance The distances between each pair of strands (measure 5) were between strands) or individual (Figure 7). also measured (Figure 7). Calculations confirm the trends previously observed. The metal-free fibril has approximately the same distance between each pair of strands. Such system has a higher flexibility With these descriptors, it is possible to see that the three on the vertical axis, as measures 2 and 4 also support, while the complexes behave similarly both in the horizontal and in the vertical horizontal axis is more compact and shows a collective movement axes (measures 1 and 2). For measure 1 (Figure 6E), the metal-free of all the strands, which corresponds to the hinge fan-like movement. fiber shows a distance of 24.64 (±5.6) Å, while Al(III) and Cu(II) However, upon metal coordination, the distance between the strands exhibit values around 25 Å, which are very similar to the 24.6 Å of differs. the ss-NMR structure. Distances of measure 2 (Figure 6F) are also First, Cu(II) ion, which binds to the NTer region, increases the similar, with the values of 19.9 (±0.3) Å for the metal-free fiber, 18.7 distance between each pair of strands, even between those bound (±0.3) Å and 18.8 (±0.2) Å for the Cu and Al bound complexes and to the same metal ion. The most remarkable is the increase in 18.3 Å for the ss-NMR structure, respectively. The other distances, the 4-His14–5-His14 distance, which indicates a disruption in the though, demonstrate the substantial differences between the metal- collective movement of the strands (Figure 7). This only happens free fiber and the metal-bound complexes. in the Cu-bound system. For Al(III), the distance between the Frontiers in Neuroscience 08 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 FIGURE 7 Average distance between His14 from each core strand to the adjacent one along 100 ns molecular dynamic (MD) simulation for metal-free fibril (dark blue), Cu(II) (light blue) and Al(III) (green). Standard deviation range from 0.1 to 0.7. Strands 3–4, 5–6, and 7–8 are bound by metal coordination in Cu(II) and Al(III) systems. FIGURE 8 Overlapping of the lowest energy structure obtained from the Gaussian accelerated molecular dynamics (GaMD) simulation of Metal-Free Fibril (A), Cu(II) (B), and Al(III) (C) against the Aβ42 model. pairs of strands is also increased, especially in strands 3 to 6, but Conclusion the collective movement is preserved. This points to the fact that, although Al(III) binding increases the compactness and stability of One of the Alzheimer’s disease main hallmarks is the formation the core region, as measures 1 to 4 support, the (NTer ) region is still of amyloid plaques, constituted by the aggregates of β-amyloid highly flexible. peptides. In this process, metal ions have been shown to play an The mentioned changes in metal coordination are also clearly important role. In the present study, an integrative computational observed if the systems are overlapped against the ss-NMR structure study has been performed to unravel the role of Cu(II) and Al(III) (Figure 8), which demonstrates that the metal-free fiber has a ion binding in the β-amyloid fibrillary structure (pdb code 2MXU). collective hinge fan-like movement, increasing the flexibility of the To do so, the binding site and the coordination sphere of the system but without its disruption. In contrast, Cu(II) leads to the metallic ions were identified with a combined protocol of BioMetAll partial dismantling of the fibrillary conformation in the (NTer ) region and Gold Software. Once binding sites were properly detected and by breaking the collective movement of the fiber. Finally, Al(III)- the tridimensional models properly set up, the complexes were bound system is more compact in the core region, though the (NTer ) submitted to an exhaustive conformational exploration through MD region is still highly flexible. and Gaussian accelerated MD, with three replicas of 1 µs each, Frontiers in Neuroscience 09 frontiersin.org Roldán-Martín et al. 10.3389/fnins.2023.1110311 achieving a total of 3 µs for the metal-free fiber, Cu(II)-bound, and Data availability statement Al(III)-bound systems. The resulting simulations were analyzed and compared between them and in front of the initial ss-NMR structure. The original contributions presented in this study are included Metal-free simulations allowed us to characterize the collective in this article/Supplementary material, further inquiries can be low-energy vibrational modes that constitute the natural breathing directed to the corresponding authors. motions of the S-shaped structure. The main one could be described as a hinge fan-like movement due to the opening–closing tendency of the (NTer ) region of the fiber. Such movement could be linked Author contributions to the dynamic process of aggregating and dismantling β-amyloid fibers. Metal-bound complexes show a differential behavior that, LR-M carried out the experiment. LR-M, J-DM, and MS wrote the interestingly, depends on the metal ion. On the one side, Cu(II) manuscript. J-DM and MS supervised the project and conceived the original idea. All authors contributed to the article and approved the binding is at the (NTer ) region with two His13 and Gln15 from submitted version. adjacent strands, at a site very close to where it happens in the monomeric species. The binding of copper ion introduces the charges in the (NTer ) region and the analyses of the simulations show that the system tends to disrupt the fibrillary structure of the most extreme Funding strands that adopt α-helix configurations, resembling the monomeric This work was supported by the projects PID2020-116861GB-I00 structure of the β-amyloid peptide. This fact supports the hypothesis and PID2020-112715GB-I00 from the Spanish Ministerio de Ciencia that copper is more prone to lead to amorphous aggregates (Huy y Educación. LR-M would like to thank Generalitat de Catalunya et al., 2016), as shown experimentally (Innocenti et al., 2010). In (grant 2020FI_B2_01000). contrast, Al(III) binding takes place in the first coil region of the S-shape, on the residues Glu22 and Asp23 from adjacent strands, which compensates for the Al(III) charge. In this case, aluminum Conflict of interest ion binding reinforces the stability of the system in the core region, even reducing the hinge movement observed in the metal-free fiber The authors declare that the research was conducted in the complex and obtaining structures with the lowest difference with absence of any commercial or financial relationships that could be respect to the ss-NMR structure. Such fact reinforces the idea that construed as a potential conflict of interest. Al(III) stabilizes neurotoxic species (Miller et al., 2012). In contrast, the larger stability of the fibril has been related to a higher aggregation rate (Thu and Li, 2022). However, mature fibril deposits are less Publisher’s note relevant than oligomers with respect to AD etiology and severity (Miller et al., 2012). All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Overall, the computational study carried out in this work shows organizations, or those of the publisher, the editors and the reviewers. that the binding of metal ions to β-amyloid fiber indeed affects its Any product that may be evaluated in this article, or claim that may dynamical behavior, though in a different manner depending on be made by its manufacturer, is not guaranteed or endorsed by the the metal ion involved: while Cu(II) leads to less organized tertiary publisher. structures than the unbound system, Al(III) retrieves the opposite behavior, with a high stable S-shaped structure. This is in line with the study of Bolognin et al. (2011), which demonstrates that while Supplementary material Cu(II) prevents the formation of fibrillary aggregates, Al(III) induces the aggregation of fibrillary oligomers. The next steps are being The Supplementary Material for this article can be found online performed to merge the influence of metal-ion coordination with the at: https://www.frontiersin.org/articles/10.3389/fnins.2023.1110311/ relevant familiar mutated forms of the β-amyloid fiber. full#supplementary-material References Alí-Torres, J., Maréchal, J. D., Rodríguez-Santiago, L., and Sodupe, M. (2011). Three Bakan, A., Meireles, L. M., and Bahar, I. (2011). ProDy: Protein dynamics inferred from dimensional models of Cu 2+-Aβ(1-16) complexes from computational approaches. theory and experiments. Bioinformatics 27, 1575–1577. doi: 10.1093/bioinformatics/ J. Am. Chem. Soc. 133, 15008–15014. doi: 10.1021/ja203407v btr168 Alí-Torres, J., Mirats, A., Maréchal, J. D., Rodríguez-Santiago, L., and Sodupe, M. Bayly, C. 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