人工GM1糖鎖クラスターへの結合に共役した アミロイドβ 40の立体構造変化/ Conformational Change of Amyloid-β 40 in Association with Binding to GM1-Glycan Cluster Yuhei Tachi1, 2, Yuko Okamoto1, Hisashi Okumura2, 3, 4 (1Nagoya University, 2Institute for Molecular Science, 3SOKENDAI (The Graduate University for Advanced Studies, 4Exploratory Research Center on Life and Living Systems) 1

Aggregates of amyloid-β (Aβ) are the causative substance of Alzheimer’s Disease (AD) 1 ~ 12 20 30 40 ・・・ QKLVFF AEDVGSNKGA IIGLMVGGVV β1 region (PDB: 2BEG) β2 region (PDB: 2BEG) Aβ-positive senile plaque [1] [1] Haas, C. and Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 8, 101-112 (2007). [2] Straub, J. E. and Thirumalai, D. Annu. Rev. Phys. Chem. 62, 437-463 (2011). Free energy landscape of Aβ fragment [2] 100 µm 2 The partially-unfolded conformations capable of protein self-assembly are rare.

1 ~ 12 20 30 40 ・・・ QKLVFF AEDVGSNKGA IIGLMVGGVV β1 region (PDB: 2BEG) β2 region (PDB: 2BEG) Aβ-positive senile plaque [1] [1] Haas, C. and Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 8, 101-112 (2007). [2] Straub, J. E. and Thirumalai, D. Annu. Rev. Phys. Chem. 62, 437-463 (2011). 100 µm β1 region β2 region 3 The partially-unfolded conformations capable of protein self-assembly are rare. Aggregates of amyloid-β (Aβ) are the causative substance of Alzheimer’s Disease (AD) Free energy landscape of Aβ fragment [2]

GM1 cluster induces the pathological aggregation of Aβ peptide GM1 containing liposome GM1 without liposome GM1 containing liposome Electron micrograph of Aβ40 solutions with liposome Monosialotetrahexosyl ganglioside (GM1) ThT fluorescence intensities : Aβ with GM1-containing liposome + : Aβ with GM1-lacking liposome : Aβ only : GM1-containing liposome only H. Hayashi, et al., J. Neurosci. 24, 4894-4902, (2004). Ceramide 4 Glycan In fact, GM1 bound to Aβ (GAβ) was experimentally found in AD human brain. 50 nm

GM1-glycan Cut the ceramide moiety Metal ions (Pd2+) S. Sato, et al., Angew. Chem. Int. Ed. 54, 8435-8439 (2016). Self-assembled GM1-glycan cluster ~5. 7 nm Interactions between Aβ40 and artificial GM1-glycan cluster are observed by NMR analyses Ceramide Glc Gal Neu GalNAc Gal’ Ligand Relative intensities of backbone NH signals of 15 N-labeled Aβ40 plotted against the amino acid sequence Aβ40 recognizes the GM1-glycan cluster using its N-terminus side. N-terminus side 5

Approach We investigate structures of the GM1-glycan cluster bound to Aβ40 by Molecular dynamics (MD) simulations Purpose ・ How Aβ40 recognizes the GM1-glycan cluster by using its N-terminus side? ・ Which conformations are adopted on the GM1-glycan cluster? 6

Computational details System1: Aβ40 + artificial GM1-glycan cluster + 31083-31103 water molecules Simulation time：1.5 µs × 9 Temperature: 300 K (experimental condition) Ensemble: NVT System2: Aβ40 + 12998-13020 water molecules Simulation time：1.0 µs × 9 Temperature: 300 K Ensemble: NVT 7

A typical 300 ns trajectory of MD simulations of Aβ40 and the GM1-glycan cluster 8

Aβ40 (Orange) GM1-glycans bound to Aβ40 (Green) Formation of GM1 glycan cluster bound to Aβ40 ・ Aβ40 binds to the GM1-glycan cluster at ~ 30 Å after ~300 ns in each MD simulation ・ Multiple GM1-glycans gradually bind to Aβ peptide ・ The average numbers converge in both cutoff distances after ~ 500 ns MD simulations Time series of distance between centers of mass of Aβ40 and the GM1-glycan cluster in each different trajectory Time series of average number of GM1- glycans bound to Aβ40 with different cutoff distances, 3.5 and 5.0 Å. 9

Importance of HHQ (13-15) regime for the GM1-glycan cluster recognition Average number of hydrogen bonds between Aβ40 and the GM1-glycan cluster (upper figure), average electrostatic interaction energy (middle figure), and van der Waals (VDW) interaction energy (lower figure) 10 Box plot of minimum distance between Aβ40 and the GM1-glycan cluster ・ The HHQ (13-15) regime closely interacts with GM1-glycan cluster. → It is consistent with the previous experimental results. ・ VDW interaction energy correlates with the approach of HHQ (13-15) region.

Histidine residues (13-14) non-specifically interact with sugar residues by stacking interactions 11 Matrix of average minimum distance between each amino acid residue and each sugar residue including Ligand (xi : minimum distance of i-th subsampled trajectory, N: total number of subsampled trajectories) It was confirmed that the imidazole group of Histidine (13-14) stack on the ring of sugar residues (right figure).

α-to-β conformational transition on GM1 cluster is the pathological aggregation pathway S. G. Itoh, et al., J. Phys. Chem. B 123, 160-169 (2019). 親水的 疎水的 GM1 (a) Schematic drawing of Aβ40 on the hydrophilic/ hydrophobic interface of the GM1 micelle. (b) The helix formation probability of Aβ40 at the hydrophilic/hydrophobic interface (b) Simulation 12 The amino acid residues of 13C signal : upfield shift : downfield shift : unchanged (a) Experiment What conformations are adopted on the GM1-glycan cluster??

We found that the GM1-glycan cluster significantly stabilizes helix structures at 31-37 residue. →A previous work was demonstrated that 31-36 residue of Aβ peptide forms helical structures on the GM1 micelle. 13 Pathological helix formation of Aβ40 on the GM1-glucan cluster

α-to-β conformational transition on GM1 cluster is the pathological aggregation pathway S. G. Itoh, et al., J. Phys. Chem. B 123, 160-169 (2019). Hydrophilic Hydrophobic GM1 (a) Schematic drawing of Aβ40 on the hydrophilic/ hydrophobic interface of the GM1 micelle. (b) The helix formation probability of Aβ40 at the hydrophilic/hydrophobic interface (b) Simulation 14 The amino acid residues of 13C signal : upfield shift : downfield shift : unchanged (a) Experiment Which conformations are adopted on the GM1-glycan cluster??

Differences of Cα distance and contacts The Cα distance between residues 38-40 and residues 27-32 increases, and simultaneously the number of Cα contacts between residues 37-40 and residues 30-33 decreases on the GM1-glycan cluster. 15 Cα distance Cα contact Amino acid residue number Amino acid residue number Amino acid residue number Distance (Å) Difference Aβ40 on the GM1-glycan cluster 1 Residue 27-32 Residue 38-40 Residue 37-40 Residue 30-33 1 （i, j：Labels of amino acids, x: Cα distance and contacts）

Propensities of each secondary structure except helix structure On the GM1 glycan cluster, propensities of turn, bend, and β-sheet structures mainly decrease instead of stabilizing helix structures. 16

17 Note that positively charged Lys28 forms salt bridges with negatively charged Neu and C-terminus. Propensities of each secondary structure except helix structure

Not only the HHQ regime, but also binding of residues 26-29 strongly correlate with the helix formation on the GM1-glycan cluster. Matrices of cross-correlation between helix formation and binding to the GM1-glycan cluster 18 This region formed helix structures more stably

Binding Lys28 to Neu plays a key role in the pathological helix formation ① Salt bridge formation between Lys28 and Neu by binding Aβ to GM1-glycans inhibits to form a salt bridge between Lys28 and C-terminus ② Helical structures are stabilized instead of turn, bend, and β-sheet structures in C-terminal side energetically. 19

Summary 1. The HHQ (13-15) regime plays a important role to recognize the GM1-glycan cluster. The GM1-glycan recognition mechanism of the HHQ segment is explained by non-specific stacking interactions between side chains of histidine residues and rings of sugar residues. 20 2. We found that Aβ40 exhibits helical structures in residues 31-37. The result suggest the GM1-glycan moieties initially trigger the pathological helix formation rather than the lipid hydrocarbon chain moieties. →It helps to design drug materials for AD and self-assembled supramole -cules at atomic level.