Disclosing the template-induced misfolding mechanism of Tau protein

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Disclosing the template-induced misfolding mechanism of Tau protein by studying the dissociation process of boundary chain from the formed Tau fibril based on the steered molecular dynamics simulation Hongli Liu, Xuewei Liu, Shuangyan Zhou, Xiaoli An, Huanxiang Liu, and Xiaojun Yao ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00732 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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ACS Chemical Neuroscience

Disclosing the template-induced misfolding mechanism of Tau protein by studying the dissociation process of boundary chain from the formed Tau fibril based on the steered molecular dynamics simulation

Hongli Liu1, Xuewei Liu2, Shuangyan Zhou1, Xiaoli An2, Huanxiang Liu1*, Xiaojun Yao2,3 1 School 2 State

of Pharmacy, Lanzhou University, Lanzhou 730000, China

Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, China

3 State

Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for

Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China

* Corresponding author Tel.: +86-931-891-5686 Fax: +86-931-891-5686 E-mail address: [email protected]

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Abstract The level of Tau aggregation into neurofibrillary tangles, including paired helical filament (PHF) and straight filament (SF), is closely associated with Alzheimer’s disease. Despite the pathological importance of misfolding and aggregation of Tau, the corresponding mechanism remains unclear. Therefore, to uncover the misfolding mechanism of Tau monomer under the induction of formed PHF and SF, in this study, the conventional molecular dynamics simulation combined with steered molecular dynamics simulation were performed to study the dissociation process of boundary chain. Interestingly, our results show that the dissociation mechanisms of boundary chain in PHF and SF are different. In PHF, the boundary chain begins to dissociate from β2 and β3 regions and ends at β8. However, in SF, it is simultaneously dissociated from β1 and β8 and ends at β5. The dissociation of boundary chain is the reverse process of template-induced misfolding of monomer. Therefore, we can deduce the misfolding mechanism of monomer under the induction of template. For PHF, β8 firstly interacts with the template by hydrophobic interaction. Then β7, β6, β5, β4 and β1 sequentially bind to the template by electrostatic and hydrophobic interactions. After β1 binds to the template, β2 and β3 very quickly bind to the template through hydrophobic interaction. For SF, β5 of monomer firstly interacts with the template by electrostatic attraction. Then β4 and β6, β3 and β7, β2 and β8 bind to the template in turn. Finally, β1 and β8 are fully bound to the template by hydrophobic interaction. The obtained results will have the vital value for understanding the earlier events during misfolding and aggregation of Tau.

Keywords:

Tau; Steered molecular dynamics simulation; Template-induced

misfolding; Alzheimer’s disease.

Introduction Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. With the increasing number of elderly people worldwide, the AD shows a rapid growth trend. 2


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For instance, the 2018 figures suggested that an estimated 5.7 million Americans have Alzheimer’s disease. By mid-century, the number of people living with AD in the United States is projected to increase up to 13.8 million.1 The exact etiology and pathogenesis of AD are still unclear, but a large number of studies have shown that the pathological features of the disease includes the appearance of senile plaques (SP) extracellular formed by β-amyloid (Aβ), and neurofibrillary tangles (NFTs) in intracellular formed by excessively phosphorylated Tau.2-7 In recent years, a variety of approaches for treating AD with Aβ as a target have been developed clinically. However, several Aβ drugs targeting different mechanisms have failed to prove their effectiveness in clinical trials.8-10 Therefore, for the treatment of AD, it is urgent to find new and more effective targets, among which Tau protein has received widespread attention. Currently, there are a considerable number of ongoing trials evaluating the ability of tau inhibitors to reduce AD progression and it is exciting that some progress has been made in their studies.11-14 NFTs are made of paired helical filament (PHF) and straight filament (SF). PHF is the main constituent of NFTs and has a unique morphology, which is composed of twisted double-helical ribbons, an imaging alternating in width between 80 Å and 200 Å with a cross-over spacing 800 Å.15,16 SF neurofibrillary tangles with a width of about 150 Å in AD, which did not show significant modulation of the width displayed by the PHF in electron micrographs.17 What’s more, in 1985, Crowther and Wischik18 first proposed that PHF is C-shaped unite, and later Crowther16 observed that SF was also a C-shaped unite. Interestingly, the hybrid filaments of SF and PHF were observed, implying that they have a similar C-shaped subunit, but differ in assembly. How are two types of cores, PHF and SF, formed? And how does it induce misfolding of monomers in two forms of cores? So far, these processes are still unclear. Since the misfolding and aggregation of Tau protein play a vital role in the development of AD, to reveal the misfolding mechanism induced by template is very important for disclosing the underlying pathogenesis of AD and designing the corresponding inhibitors. Fortunately, in 2017, Fitzpatrick et al.19 determined the high-resolution structures of Tau filaments, including PHF and SF, from the brains of individuals with 3



AD by the cryo-electron microscopy (cryo-EM). Consistent with the findings from Crowther,16 both PHF and SF are composed of two protofilaments with the C-shaped subunits and the core structure composed primarily of R3 and R4 regions. The 3D structure of PHF and SF of Tau can provide a good start to study the mechanism of formation of two cores and the misfolding mechanism of monomer under the induction of template. However, the cryo-EM structure can only provide static information. How is the core formed and how does the monomer misfold under the induction of core? These dynamics information are difficult to be revealed through the existing experimental methods. Compared to the conventional experimental methods, molecular dynamics (MD) simulation method has unique advantages in studying the structural changes of proteins.20 It has been widely used in the study of misfolding and aggregation of proteins.21-23 However, whether the molecular dynamics simulation methods can uncover the correct protein misfolding mechanism depends on several limitations, including the size of the studied system, the used simulation method, the time scale of simulation, and the analysis method of the obtained trajectory. Full-length Tau protein contains 441 residues, and is quite difficult to directly study the misfolding of monomer under the induction of template by applying molecular dynamics simulation methods. Here, in order to reveal the misfolding mechanism of Tau protein under the induction of template, the dissociation process of the boundary chains from the formed fibril were investigated by steered molecular dynamics (SMD). In SMD, the timedependent external forces are applied to the biomolecular systems along the reaction coordinates to study the dynamics of binding-unbinding events. The simulations can reveal the details of molecular interactions in the course of unbinding or unfolding, thereby providing important information about molecular mechanisms underlying these processes. The advantage of SMD over conventional MD is that relatively large molecular conformational changes can be induced on the nanosecond time scale, which is difficult to be achieved in conventional MD simulation.24,25 Furthermore, the residue interaction network (RIN) analysis and dynamical network analysis were also calculated to show the detailed interaction changes between the boundary chain and the 4


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core during the dissociation process, further uncover the dissociation mechanism of boundary chain.

Results The overall structural characteristics of PHF and SF The fibril of Tau protein mainly contains two forms, PHF and SF.19 In order to investigate the interaction features of inter-chains in PHF and SF structures and provide the basis for the dissociation mechanism study of the boundary chain, the conventional MD simulation was performed firstly on two types of fibrils. To monitor whether the simulations are up to converge, the root mean square deviation (RMSD) of protein backbone atoms from the first structure of MD simulations trajectories were calculated. Figure 1A indicates that after 120 ns, both systems are converged. From the Figure 1A, it can be seen that the RMSD value of SF is maintained at around 4 Å and has larger fluctuations than PHF system (about 2 Å), indicating that the SF system is more flexible as a whole and the PHF system is relatively stable. Furthermore, the distribution of the radius of gyration (Rg) shown in Figure 1B indicates that the distribution of Rg of SF is larger than PHF, showing that the overall structure of PHF pentamer is more compact than the SF.

Figure 1. Structural characteristics of PHF and SF were calculated by the conventional molecular dynamics simulations. (A) Time evolution of the RMSD of protein backbone atoms for PHF (black line) and SF (red line). (B) The distribution of radius of gyration 5



of PHF and SF model.

The interaction map between boundary chain and its neighbour In order to study the interaction between boundary chains and the adjacent chains of PHF and SF systems, the dynamic cross-correlation map (DCCM) analysis, dynamical network analysis and hydrogen bond (H-bond) interaction analysis were performed. Since the dissociation of the formed fibril or further growth of fibril begins from the boundary peptide, understanding the interaction between the boundary peptide and its neighbour is very important to understand the dissociation and growth mechanism of fibril. For convenience, five chains of pentamer are named A, B, C, D, and E respectively, as shown in Figure 2.

Figure 2. The cartoon structure of Tau protein fibril (A) and amino acid sequence (B) of Tau protein.

Figure 3 shows the dynamic cross-correlation map between the A and B chains or D and E chains calculated from the last 50 ns of the conventional molecular dynamics simulation. Highly positive regions (red) represent the strong correlations between residues, where negative regions (dark blue) refer to the strong anti-correlation motions of specific residues. The diagonal regions describe the interaction between A and B chains or D and E chains. For PHF system, Figure 3A and 3B are inter-chain interactions of AB chains and DE chains, respectively. Overall, the boundary chains on both sides have strong interactions with their adjacent chains. But at the end part of β2 and the beginning part of β3 of AB chains (ellipse region of Figure 3A), the inter-chain 6


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interaction is very weak, while it is stronger at DE chains. Therefore, the A chain in PHF system may be more easily dissociated than E chain. For SF system, Figure 3C shows that the inter-chain interaction of AB chains is strongest in the section of β5 and the beginning part of β6 (rectangle region of Figure 3C) and then weakens from this region to both sides. As shown in Figure 3D, the inter-chain interactions in DE chains are similar to that in AB chains. In other words, two sides of SF system have similar environment and may have similar dissociation trend.

Figure 3. The calculated dynamics cross-correlation map between the A and B chains or D and E chains from the last 50 ns conventional molecular dynamics. The degree of correlation and anti-correlation are corresponding to the color bar. The ellipse represents the end part of β2 and the beginning part of β3, the rectangle represents β5 and the beginning part of β6. (A) Chain A and Chain B in PHF. (B) Chain D and Chain E in PHF. (C) Chain A and Chain B in SF. (D) Chain D and Chain E in SF. The dynamical network analysis is a useful approach to study potentially 7



important interaction residues. To further investigate the inter-chain interactions in PHF and SF, the dynamical network analysis was performed and the corresponding results were given in Figure 4. Dynamical network analysis can divide the network into subnetworks with different colors, depending on the motion characteristics of nodes. In this figure, the different color domains in PHF and SF represent the different network communities. The nodes belonging to the same community are more strongly related to each other and weaker to other nodes in the network. For PHF system, from Figure 4A, there is strong interaction between the chains, especially in the β5 region. However, by comparing the interaction between two boundary chains and their adjacent chains, there is an obviously difference in the end part of β2 and Loopβ2-3 region. The inter-chain interaction between AB chains in this region is significantly weaker than that between DE chains. As shown in Figure 4A, the amino acid at position 323 of AB chains have no inter-chain interaction, and the inter-chain interactions of amino acids at positions 321 and 322 are much weaker than those of DE chain. Therefore, the A chain with the weakest inter-chain interaction may be easier to dissociate, which is consistent with the results of DCCM analysis. For SF system, Figure 4B shows that the interaction between the chains in β5 region is the strongest, while the remaining regions are weak and relatively similar. Overall, there is no difference between the A and E chain in the SF system. From the comparison, we can conclude that the A chain is the easiest to be dissociated from the formed fibril for PHF system, while for SF system, both A and E chains are likely to be dissociated.

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Figure 4. Dynamical network analysis in PHF (A) and SF (B) models. The different color domains of PHF and SF represent different network communities. The residues are represented by nodes, and the line between nodes represents edges. The thickness of line indicates the strength of interaction.

To further investigate the inter-chain interactions in more detail, the inter-chain backbone hydrogen bonds were analyzed. In Figure 5, red and green represent H-bond occupancy greater than 90% and 80%, respectively. For PHF system, as shown in Figure 5A, there are fewer inter-chain backbone H-bonds distributed in the end part of β2 and β3 regions of AB chains, indicating that the H-bonds interaction is weaker in this region. While for DE chains, shown in Figure 5B, backbone H-bonds interaction is stronger than that in AB chains, especially in β2 and β3 regions. Thus, the A chain is more easily dissociated than E chain, which is consistent with the results of DCCM and dynamical network analysis. For SF system, Figure 5C and 5D show that the distribution of backbone H-bonds between the AB and DE chains is relatively similar, suggesting SF pentamer has the same environment on both sides, which is consistent with the DCCM and dynamical network analysis. From the above analyses, we can infer that the β2 and β3 regions of AB chain of PHF system have the weakest inter9



chain interaction and are most likely to dissociate from this region. For SF system, the environment of the boundary chain is similar. Since the inter-chain interaction of β5 region is strong, it is most likely to be dissociated from β1 and β8.

Figure 5. Inter-chain backbone hydrogen bonds analysis. Red and green represent the hydrogen bonds occupying more than 90% and 80% respectively. (A) Chain A and Chain B in PHF. (B) Chain D and Chain E in PHF. (C) Chain A and Chain B in SF. (D) Chain D and Chain E in SF.

Based on the DCCM, dynamical network analysis and backbone H-bond analysis, it can be deduced that the boundary chain of PHF is most likely to dissociate from β2 and β3 region of the A chain. In SF system, there are similar interaction environments on both sides of the boundary chains, and both sides are the potential sites to dissociate. To study the dissociation mechanism of boundary chain and then uncover the misfolding mechanism of monomer in the induction of template or fibril growth mechanism, the SMD simulation was used to pull the A chain of PHF from the formed 10


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fibril, as well as A and E chains of SF.

The dissociation mechanism of PHF boundary chain Based on the analyses of the conventional MD, in PHF model, the inter-chain interaction between AB chains is weaker relatively than that between DE chains. Thus, the A chain should be easier to dissociate from the formed fibril. To investigate its dissociation path of boundary chain, SMD simulation was performed by pulling the A chain along Z-axis and using the distance between the mass center of A chain and the mass center of other section of the fibril as the reaction coordinate. To ensure the reliability of results, three trajectories with spring constant of 50 kcal/(mol·Å2) and velocity of 0.013 Å/ps were repeated. All three trajectories meet the requirements of hard spring and the fluctuation of force curve had a similar trend (as shown in Supplementary Figure S1). Therefore, one of them is used for the following analysis. First of all, the changes of secondary structure of A chain during the stretching were monitored and shown in Figure 6A. From this figure, the secondary structure of A chain gradually changes from β-sheet to coil structure at different stages. Firstly, β and β3 rapidly transform into a coil structure, followed by the transition of β1 and β4, β5, β6, β7, and β8 regions. Finally, the A chain completely changed into a coil structure at 8 ns, which is consistent with the experimental reports that Tau protein is a disordered structure naturally. What’s more, the variation curve of force versus reaction coordinate for two systems was shown in Figure 7. From Figure 7A, according to the change of force curve and secondary structure analysis, the dissociation of A chain of PHF can be divided into five stages. Firstly, the earliest dissociating region is β2 and β3, followed by the dissociation of peptides β1, β4 and the beginning part of β5. After that, the end part of β5 and Loopβ5-6 regions begin to dissociate. This is followed by the dissociation of β6 and β7. The dissociations of these two stages have been observed to be slower because of stronger inter-chain interaction. Finally, β8 dissociates from the formed fibril.

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Figure 6. Time evolutions of secondary structure of Chain A of PHF model (A) and SF model (B) and Chain E of SF model (C).

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Figure 7. The force profiles over the reaction coordinate for the PHF and SF systems when the different boundary chains are pulled. I, II, III, IV, and V represent the five 13



stages of dissociation, respectively. The arrow points to the representative conformation of each stage, where the red part in the conformation indicates the region of dissociation at that stage. (A) Chain A of PHF. (B) Chain A of SF. (C) Chain E of SF.

Under the template induction, what is the driving force of disordered monomer to transform into a misfolded conformation? To answer this question, we analyzed the changes of van der Waals (vdw) and electrostatic interactions of boundary chain with other sections, and the obtained results are shown in Figure 8. The van der Waals and electrostatic interactions in gas phase was calculated based on the calculation formula of the van der Waals (vdw) and electrostatic forces in the amber force field.26 In Figure 8, red, purple, green, magenta, and black respectively represent the five stages of dissociation. The straight line represents the electrostatic interaction and the dashed line represents the van der Waal interaction. By monitoring the changes of vdw and electrostatic interactions (Figure 8A), vdw interaction is mainly destroyed in the first stage of dissociation, and then both electrostatic and vdw interactions are disrupted to the similar extent during the next three stages in the middle of dissociation. During the final dissociation stage, the vdw interaction is mainly disrupted. The dissociation of the boundary chain is the reverse process of monomer misfolding under the induction of template. Therefore, in the process of monomer misfolding of PHF system, the hydrophobic interactions firstly drive the binding of monomer to the template, and then the electrostatic and hydrophobic interactions together stabilize the binding of monomer to the template, and the final stage of binding is mainly dependent on the hydrophobic interactions.

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Figure 8. The time dependence of the interaction energy between boundary chain and template for PHF (A) and SF (B) systems. Different colors represent different stages. The colors of five stages are red, purple, green, magenta, and black in order. In addition, the straight line represents electrostatic interaction and the dashed line represents van der Waal interaction. To show the detailed changes of interaction between boundary chain and formed fibril, the residue interaction network analysis was performed. The dissociation of boundary chain can be divided into five stages, and the first conformation (2.0 ps, 0.32 ns, 1.74 ns, 3.50 ns, and 5.45 ns) in each stage was extracted as the representative conformation to construct residue interaction networks by using the Ring web server27 and Cytoscape28 software. Figure 9A shows the dynamics change of residue interaction networks between the pulled A chain and B chain in PHF model during SMD simulation. Figures 9A (a), (b), and (c) show the changes in vdw interaction, H-bond, and π-π stacking interaction during dissociation, respectively. In Figure 9A, pink and lavender spheres represent the residues of the A and B chain, respectively. The cyan sphere represents the residues that are about to dissociate at the current stage. The red sphere represents the residues with the increased interaction, compared to the previous stage. The magenta sphere represents the residues with the increased interactions compared to the previous stage but to be dissociated compared to the next stage. The green, blue, and magenta dash lines represent the vdw interaction, H-bond, and π-π stacking, respectively. 15



The first stage is the dissociation of β2 and β3. During this process, the vdw interactions of K317, V318, K321, N327, H329, H330, and P332 with B chain, the Hbonds of D314, S316, V318, S320, C322, G323, G326, I328, and H330 with B chain are destroyed. In addition, two π-π stacking interactions formed by histidine (H329 and H330) on β3 with B chain were also destroyed. The second stage is the dissociation of β1, β4 and the beginning part of β5. During this process, Y310 located on β1 is a key amino acid. The vdw interaction, H-bond, and π-π stacking interaction formed by Y310 and the amino acids of B chain are destroyed. In addition, vdw interactions formed by Q307, I308, V313, Q336, S341, L344, and D345 with residues of B chain and the Hbonds formed by V306, I308, D314, V339, S341, and E342 with the residues of the B chain are also destroyed. Figure 9A-(a)-II shows that due to the dissociation of the β1 and β4 region, the part of residues of A chain (D345, K347, Q351, G355, I360, K369, I371, and K375) in the β5 to β8 region are locally adjusted by increasing vdw interactions with the residues of B chain to stabilize the entire protein. The third stage is the dissociation of section K347-R349 (including the end part of β5 and Loop β5-6 region). In this process, the vdw interactions formed by K347 and R349 of A chain with D348 and R349 of the B chain (Figure-9A-(a)-III) and H-bonds formed by R349 of A chain and D348 of the B chain (Figure-9A-(b)-III) are disrupted. The fourth stage is the dissociation of the β6 and β7. In this period, a large number of vdw interactions, including V350, S352, I354, S356, L357, T361, and G366 of A chain with B chain are destroyed. The last stage is the dissociation of β8, where H374 and F378 are the two vital amino acids. The vdw interactions, H-bonds, and π-π stacking they formed with B chain were destroyed.

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Figure 9. The residue interaction network analysis between Chain A and Chain B of PHF (A) and SF (B). The edges are colored according to their interaction type. The green, blue, and magenta dash lines represent the vdw, H-bond, and π-π stacking 18


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interactions, respectively. Pink and lavender sphere or hexagonal represent the residues of the A and B chain, respectively. The cyan sphere or hexagonal represents the residues that are about to dissociate at the current stage. The red sphere or hexagonal represents the residues with the increased interaction, compared to the previous stage. The magenta sphere or hexagonal represents the residues with the increased interactions compared to the previous stage but to be dissociated compared to the next stage.

The dissociation mechanism of boundary chain in the SF model The results of the conventional MD show that the environment on both sides of the SF model is similar, so SMD was performed on both A and E chains of SF system to investigate whether the dissociation paths on both sides are same. Similarly, under spring constant of 50 kcal/(mol·Å2) and velocity of 0.010 Å/ps, three trajectories for the dissociation of the A and E chains were repeated and all of them have reached the hard spring condition and the changes in force curve are similar (as shown in Supplementary Figure S1). Thus, one of three trajectories was selected for the following analysis. From the secondary structure analysis shown in Figure 6B and 6C, it can be seen that both A and E chains have similar dissociation paths with a symmetrical dissociation from β1 and β8 and finally ending at β5. According to the change of secondary structure and force curve (Figure 7B and 7C), the dissociation of A and E chains can be divided into five stages. The representative conformations (shown in Figure 7B and 7C) extracted from each stage show that the A and E chains have the same dissociation pathway, which dissociates from β1 and β8 and then develop symmetrically towards β5. Then, only the dissociation process of A chain was analyzed further. In order to investigate the driving force of the disordered monomer transforming into a misfolded conformation under template induction, the van der Waals (vdw) and electrostatic interactions between A and B chains were analyzed and shown in Figure 8B. Figure 8B shows that the first stage of dissociation primarily disrupts the vdw interaction. From the second to fourth stages of dissociation, the vdw interaction gradually decreases, while the electrostatic interaction fluctuates greatly. The last stage 19



mainly breaks the electrostatic interaction between the A and B chains. The dissociation of the boundary chain is the reverse process of template induced misfolding of the monomer. Therefore, under the induction of template, the monomer is first attracted to the template by the electrostatic interaction, and then the van der Waals and electrostatic interaction synergistically stabilize the binding of the monomer to the template, which is different from the template induced misfolding mechanism in PHF system. The further residue interaction network analysis (shown in Figure 9B) shows the detailed interaction changes during the dissociation process of monomer from the formed fibril. The first stage is the dissociation of β1 and part of β8, which destroys a large number of H-bonds, including Q307, V309, K311, K375, and T377, part of vdw interactions, including I308, P312, and V313, the π-π stacking formed by Y310 and F378 with residues of B chain. The second stage is the dissociation of β2 and β8, which mainly destroys a large number of H-bonds, including L315, T319, K321, P364, K369, and I371 (Figure 9B-(b)-II). In addition, the vdw interaction formed by L315, T319, and P364 with the residues of B chain were also disrupted. Interestingly, P332 of β4, as well as S316, V318 (located at β2), N368 and T373 (located at β8), which are about to dissociate, form the increased vdw interaction compared to the previous stage. It indicates that hydrophobic interaction plays a key role in the stable binding of monomer to the template. The third stage is the dissociation of β3 and the end part of β7, in which H330 located at β3 and H362 located at β7 are the vital amino acids, and their vdw, Hbonds and π-π stacking interactions formed with B chain are all destroyed. As the disappearing of these interactions and the disruption of the secondary structure of Chain A, the vdw interactions between residues of A chain (I328 of β3 and L357 of β5) and residues of B chain (V363, T361 and L357) increase to stabilize the overall structure (as shown in Figure 9B-(a)-Ⅲ). The fourth stage is the dissociation of β4, the end part of β6 and the beginning part of β7, which destroys the vdw interactions formed by E342, Q351, and S356 of Chain A and chain B, the H-bonds formed by Q336, S341, E342, Q351, S356, and D358 of Chain A and Chain B, and the π-π stacking interaction formed by F346 of Chain A and F346 of Chain B. In the last stage, the inter-chain Hbonds (K343, D345, and K347, Figure 9B-(b)-V) and vdw interactions (L344, D348, 20


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and V350, Figure 9B-(a)-V) in the β5 and the beginning part of β6 region are destroyed.

Discussion and conclusion Based on the SMD simulation and detailed analyses, this study discloses the dissociation mechanism of boundary chain from PHF and SF fibrils. As shown in Figure 10A, PHF easily dissociates from the β2 and β3 regions of A chain, followed by β1 and β4, then dissociates sequentially along the direction of β5, β6, β7, and β8. Reversely, under the induction of template, β8 section of monomer is most easily to interact with the formed core firstly. According to the analyses of the residue interaction network and energy change, the monomer first binds to the template drived by hydrophobic interaction between β8 section of monomer and fibril. Then, β7, β6, β5, β4 and β1 sequentially bind to the template. Among them, since β4, β5 and β6 are arranged in a triangular fashion to form a β-helix structure, they need to undergo a conformational transition, so the binding process is slow. In these several processes of binding, both the electrostatic interaction and hydrophobic interaction are important for the binding of monomer and template. After β1 binds to the template, β2 and β3 rapidly bind to it. Thereby, for PHF system, the monomer firstly binds to the template mainly by hydrophobic interaction, and then electrostatic interaction and hydrophobic interaction together facilitate the stable binding of the monomer to the template.

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Figure 10. The schematic representation of dissociation mechanism of boundary chain and the deduced misfolding mechanism of monomer under the induction of template in PHF (A) and SF (B) models. I represents the initial state, and II, III, IV, V, and VI represent five states of dissociation. The eight colors blue, slate, lime, green, forest, yellow, orange, and red represent the corresponding eight regions β1, β2, β3, β4, β5, β6, β7, and β8, respectively.

For SF system, the dissociation paths of two side boundary chains are basically same and then only the dissociation mechanism of A chain was explored in details. It can be seen from Figure 10B, the dissociation of A chain starts from β1 and β8 simultaneously, and then spreads symmetrically through β2, β3 and β7, β4 and β6 to β5. The dissociation of boundary chain is the reverse process of template-induced monomer misfolding of Tau protein. Therefore, according to the dissociation path and energy analysis, we can deduce the misfolding mechanism of monomer under the induction of template. In the induction of SF fibril, β5 of monomer initially interacts 22


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with the template by electrostatic attraction. Then β4 and β6 bind slowly to the template. Since β4, β5 and β6 are arranged in a triangular fashion to form a β-helix structure, the binding of β4 and β6 with the template needs to undergo conformational changes and some adjustments. After this, β3, β7 and β2, β8 bind to the template. In these processes, both the electrostatic interaction and hydrophobic interaction play important roles. In the final stage, β1 and β8 are fully bound to the template. Overall, for the SF system, the monomer first binds to the template by electrostatic attraction, and then the electrostatic and hydrophobic interactions together drive the stable binding of the monomer to the template, which is different from the PHF system. Above all, this study can provide the valuable information for understanding the Tau protein misfolding and aggregation mechanism.

Methods The preparation of simulation systems In this work, the initial 3D coordinates of PHF and SF were obtained from the RCSB Protein Data Bank29 (PDB ID: 5O3L and 5O3T). In two cryo-EM structures, each consists of two pentamers and one of two pentamers was extracted to study the dissociation process of the boundary peptide from fibril. PHF and SF are two forms of NFTs that share a similar C-shaped subunit but differ in assembly. By superimposing the monomer of the extracted 3D structures of PHF and SF models (as shown in Supplementary Figure S2), the RMSD of their backbone atoms in PHF and SF systems was found to be 1.4 Å. Their difference is mainly located in β1 and β-helix regions including β4, β5 and β6. Firstly, the stability of PHF and SF pentamers and inter-chain interactions were investigated by the conventional molecular dynamics simulation, and then the boundary chains were stretched by the steered molecular dynamics simulation to further study the dissociation mechanism of boundary chain. The standard ff14SB force field parameter30 was used to describe protein. All systems were immersed into a cubic box of TIP3P31 water with at least 12 Å distance around the protein. To maintain the electro-neutrality of each system, chloride ions were added and randomly arranged 23



in the solvent.

The conventional molecular dynamics simulation The amber 14 software package32 was employed for all the simulations, including the conventional molecular dynamics simulation and steered molecular dynamics simulation. Two systems were minimized using a steepest decent method followed by conjugate gradient method, and then gradually heated from 0 to 310 K in the canonical (NVT) ensemble with the protein constrained by 5.0 kcal/(mol·Å2). Langevin thermostat with a coupling coefficient of 2.0 ps-1 was used to control the temperature. SHAKE algorithm33 was used to restrain the bond lengths involving hydrogen atoms. All equilibration and later stages were carried out in the isothermal isobaric (NPT) ensemble. Finally, PHF and SF systems were simulated for 200 ns and the time step was chosen to be 2 fs. The criterion for hydrogen bond was defined as a cutoff of 120° for angel and cutoff of 3.5 Å for distance. The STRIDE algorithm34 was used to assign secondary structure.

Steered molecular dynamics simulation Steered molecular dynamics simulation (SMD)35 is an extended MD simulation method mimicking the principle of the atomic force microscopy (AFM). SMD employed the time-dependent external forces to the ligand or part of the region in the simulated biological system to allow it to dissociate or undergo a conformational change. Generally, there are three ways to introduce the external forces, including harmonic potential, surface tension and torsion. SMD simulation with the external force introduced by harmonic potential consists of two types: constant-velocity and constantforce. In this study, the constant-velocity SMD was used, and its basic principle is as follows. Firstly, the reaction coordinate and the pulled atoms need to be defined, and then a moving spring is used to induce the motion along the reaction coordinate. The free end of spring is linked to the dummy atom, which moves at a constant speed, while the pulled atoms attached to the other end of the spring is affected by the steering force. The applied force is determined by the pulling velocity and spring constant, which 24


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satisfies Hooke’s law: F(t) = 2k(vt ― x(t)) where k is the spring constant, v is the pulling velocity, and x(t) is the position of the pulled monomer at time t. In SMD simulation, it can not only obtain the changing curve of force with time, but also can record the dynamic change information of the system during the whole simulation process. Through the analysis of force curve combined with the structural information obtained from simulation, the interaction between protein and protein or protein and ligand, and the conformational change of protein can be qualitatively analyzed.36-41Compared with the conventional MD, it enables the biochemical processes that originally occurred in the microsecond to second time scale to be simulated in the nanosecond scale and dynamically reproduces the binding or dissociation processes between protein and protein or protein and ligand that cannot be provided by the conventional MD. Based on the above features of SMD, it is perfectly suited to study the boundary chain dissociation process. In this work, the final structure of PHF and SF extracted from the conventional MD simulation was used as initial structure for SMD simulation. The boundary chains in PHF (A chain) and SF (A and E chain) were pulled away from the core structure along the Z-axis by using the distance between the mass center of boundary chains and mass center of other section of the fibril as the reaction coordinate. The pulling velocity and the spring constant were identified using the following stepwise optimization. The spring constant value were set to 30, 40 and 50 kcal/(mol·Å2), while keep v fixed at 0.009 Å/ps. The results show that the stiff spring approximation was satisfied when a spring constant was 50 kcal/(mol·Å2) (shown in Figure S3). The distance between monomer and template is greater than 20 Å as the criterion of complete dissociation. By setting k as 50 kcal/(mol·Å2), different pulling velocity of 0.010 Å/ps, 0.011 Å/ps, 0.012 Å/ps, and 0.013 Å/ps were performed. It is found that A chain of PHF system is completely dissociated at v=0.013 Å/ps and two boundary chains of SF system are completely dissociated at v=0.010 Å/ps. At last, 50 kcal/(mol·Å2) for k and 0.013 Å/ps for v were selected for the A chain of PHF, and k and v were set as 50 kcal/(mol·Å2) and 0.010 Å/ps for the A and E chains of SF as the following SMD simulations 25



parameters. To obtain reliable results, three

parallel 10ns trajectories were performed

with different random seeds.

Residue interaction network calculation The residue interaction network (RIN) is constructed from the three-dimensional structure of protein to generate a two-dimensional network node graph. It simplifies and extracts the valuable information by applying the network theory to protein structures. RIN can identify the key residues as well as different interactions. Its basic principle is to identify the interacting residue-residue pairs based on the all-atom distance measurements and then characterize the contacts according to the identified specific interaction types.27 RIN is widely used to study the relationship between the structure and function of proteins, including the folding/unfolding of proteins, the effects of amino acid substitutions, and the binding of ligand and proteins.42-44 In the network, the node represents amino acids and the edge corresponds to the non-covalent interactions between the residues (e.g. van der Waals, H-bond, salt-bridge and π-π stacking interactions). In this work, the representative conformations of different stages in the dissociation process of each system were extracted, and then, Ring web server27 was used to generate the network and visualized in Cytoscape28 software. The generated networks were used to analyze the interactions between residues.

Dynamical network analysis The dynamical network analysis is a useful method to study the residue-residue interactions based on the coupled motions between the pairs of residues over a period of simulation time. It can be used to identify the correlated motions between different domains of a protein or inter-protein, and explore the interaction mode between the ligand and protein.45-47 In the process of building a dynamic network, two nodes are considered connected if the heavy atoms are within 4.5 Å of each other for at least 75% of the frames analyzed. Each edge was weighted by W𝑖𝑗 = ―log (|𝐶𝑖𝑗|) 26


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where Wij is the weight, and Cij is the correlation value of two end nodes. The GirvanNewman algorithm48 was used to divide the network map into the communities of highly intra-connected but loosely inter-connected node. In this work, to study the change of interactions between the residues for the PHF and SF systems, the NetworkView plugin49,50 in VMD51 was employed to construct the dynamical network by using data from the final 50 ns of 200 ns trajectories of the conventional molecular dynamics simulations.

Support information: Supplementary information includes the force profiles over the reaction coordinate for the parallel trajectories of the studies systems, the structural comparison of the two protofilament cores, and end-to-end distance and the constrained distance relationship for PHF and SF systems.

AUTHOR INFORMATION Corresponding Author *Tel.: +86--931-891-5686 Fax: +86-931-891-5686 E-mail address: [email protected]

ORCID Huanxiang Liu: 0000-0002-9284-3667

Author Contributions H.L. Liu, H.X. Liu and X.J. Yao conceived and designed the experiments. H.L. Liu, X.W. Liu, S.Y. Zhou, and X.L. An performed the experiment and analyzed the data. H.L. Liu, H.X. Liu and X.J. Yao wrote and reviewed the manuscript.

Funding This work was supported by the National Natural Science Foundation of China (Grant 27



No. 21675070) and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2017-k24).

Notes The authors declare no competing financial interests.

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Misfolding mechanism of Tau protein induced by template. 80x39mm (300 x 300 DPI)


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Disclosing the template-induced misfolding mechanism of Tau protein

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