Dimerization and Structural Stability of Amyloid Precursor Proteins

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The Dimerization and Structural Stability of Amyloid Precursor Proteins Affected by the Membrane Microenvironments Fude Sun, Long Chen, Peng Wei, Mengya Chai, Xiufang Ding, Lida Xu, and Shi-Zhong Luo J. Chem. Inf. Model., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 5, 2017

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Journal of Chemical Information and Modeling

The Dimerization and Structural Stability of Amyloid Precursor Proteins Affected by the Membrane Microenvironments Fude Sun, Long Chen, Peng Wei, Mengya Chai, Xiufang. Ding, Lida Xu*, and Shi-Zhong Luo* Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.

ABSTRACT: The lipid raft microenvironment is implicated in the generation of the pathological amyloid-β (Aβ) species in amyloid precursor protein (APP) that is associated with neurodegenerative diseases. Evidence shows that APP forms a transmembrane homodimer with changeable structures as a function of the membrane compositions. However, the molecular responsibility of the dimerization and structural alteration for the amyloidogenic process in segregated membranes remains largely unclear. Here, we performed multiple coarse grained (CG) simulations to explore the behavioral preference of the transmembrane domain of APP (called C99) that is affected by the lipid raft microenvironment. The results showed that C99 was anchored at the boundary of the lipid raft relying on the conserved hydrophobic motif of V710xxA713xxxV717xxxV721. Moreover, the dimerization of C99 was greatly destabilized by the lipid raft, which led to a susceptible switching packing conformation. The molecular driving forces were derived from the combined regulation of the saturated lipids and cholesterols rather than from the simple binding competition of cholesterol in the C99 dimerization. The molecular details of the differential dimerization in the raft-forming and bulk fluid bilayer environments were compared, and the structural information was helpful for further understanding the enzymolysis responsiveness of APP.

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INTRODUCTION Alzheimer’s disease (AD) is a globally occurring neurodegenerative disease that is clinically characterized by a progressive memory loss and behavioral disorder 1-2. The pathogenesis of AD is largely associated with the excessive production of cytotoxic Aβ forms derived from APP that consists of a single transmembrane (TM) span 3. Numerous studies have shown that the TM domains of APP form homodimers in the native membrane environment 4-5. However, to date, no consensus has been reached regarding its dimeric modes. Two NMR structures in the left-handed and right-handed conformations of the APP TM homodimers in the micelle environment have been reported 6-7. The results mostly indicate that the packing motivation is driven by the G700xxxG704xxxG708 zipper as the central interface 8-9, while the adjacent motif of G709xxxA713 on the opposite side also plays an important role in APP dimerization 6, 10. Moreover, evidence indicates that the relative positions of the G700xxxG704xxxG708 motifs and the TM structure in APP are able to change according to the bilayer compositions 11-13. It is known that an elevated cholesterol (CHOL) level in neuronal membranes leads to increased Aβ production, and the underlying molecular mechanisms of the binding specificity 14-15, association quantization 8 and Aβ generation 16-17 have been studied extensively. It is intriguing that CHOL can convertibly bind the different packing interfaces during APP-TM dimerization. Meanwhile, the stability of the dimer is affected by unsaturated lipids 18. More recently, studies have confirmed that the release of Aβ peptides is elevated when APP dimerization is suppressed by CHOL 19-20. In addition, evidence has also revealed a preference for Aβ clustering at the CHOL enriched domains. Therefore, the dimerization of APPs involves structural stability, deformation and even Aβ generation, which are mediated by the membrane composition. In fact, the eukaryotic plasma membrane contains multiple species of lipids and, therefore, exhibits coexisting phases due to their lateral heterogeneity 21-22. Lipid rafts are bilayer compartments enriched in CHOL and saturated lipids and play an important role in accommodating many trafficking proteins 23-25. Many studies have shown that the enhanced accessibility of APP to lipid rafts in which β- and γ-secretases are likely to be associated can facilitate amyloidogenic processing 16, 26-30. Due to the advances in biotechnology, insights into the lipid rafts can be obtained using biophysical approaches 31-32. However, high-resolution perspectives of the nanoscale substructure remain difficult to obtain using current experimental conditions 33-34. In recent years, molecular dynamic simulations, particularly the Martini coarse-grained (CG) model 35-38, have been extensively used, relying on its reliable molecular resolution and efficient computation speed. To date, many simulation works have been conducted focusing on the interaction between the APP/Aβ substrates and bilayer mixtures 17, 39-40. However, almost all studies involving APP/membrane associations have used simple lipid models without a phase segregation 8-9, 12, 18 and are, therefore, limited in providing detailed systematic information regarding the priority/transition of the APP conformation in a natural membrane containing distinct subdomains. Therefore, exploring the molecular basis of the dimerization and structural alteration of APP in a realistic membrane environment is important for deciphering its relevance to AD pathogenesis. Based on existing knowledge regarding the phase-coexisting bilayer in various biological processes 41-44, the localization, dimerization ability and structural preference of APP in the raft-forming membranes were investigated using extended CG simulations in this study. The results systematically demonstrated the influence of the lipid rafts on APP in molecular details, which revealed the structural responsibility of its enzymolysis process. Information regarding the mechanism by which APP is modulated by the lipid raft can expand our understanding of the autoregulation and bioactivity response of TM proteins in the natural phasecoexisting membrane. 2


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METHODS CG model set-up. All simulations were performed using the Gromacs-4.6.3 package 45. The Martini CG model, in which four atoms plus the associated hydrogens (except for the cyclic structure) were integrated into one bead, was employed to study the peptide structure and dimerization on long-term scales 46-48. The atomistic structure of C99 was obtained from the NMR database (PDB code: 2LLM) and transferred into the CG model by martinize.py script according to the existing secondary structure information. A 15×15×8 nm3 simulation box containing 324 dipalmitoyl-phosphatidylcholine (DPPC), 180 diarachidonicphosphatidylcholine (DAPC), 216 CHOL and 160 sphingomyelin (DPSM) was constructed to imitate the raft-forming lipid bilayers 41, 49 (Figure 1A). A single-phase bilayer model consisting of 186 DPPC lipids was applied to mimic the bulk fluid bilayer environment, which has been used extensively in previous simulation studies 50-51. The lipid bilayers were solvated by CG water molecules, and counter ions were added to the systems to maintain a neutral system. The peptide monomer was perpendicularly inserted into the center of the planar membrane. For systems involving peptide dimerization, the two monomers were initially separated by a distance of 55 Å, which was ensured to exclude interference from the non-bonded interactions at the start of the simulation. Note that the C99 used here (sequence: 685 726 Q KLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK ) did not consist of a cytoplasmic tag. The sequence covered the basic fragment of enzymolysis that was responsible for γ-secretase (Figure 1A) 16, 52 . Mutants of C99 were generated using Pymol software 53. Four independent replicates were conducted for the analysis, unless indicated otherwise. CG simulation details. All constructed systems were energy minimized using the steepest descent method to exclude unnecessary atom overlapping. NPT equilibrations of 50 ns with a time constant of 1 ps were conducted at 323 K for the pure DPPC bilayer models (to maintain a fluid phase) and295 K for the raftforming bilayers 54-55. The bilayer components were semi-isotropically coupled to a heating bath using Berendsen 56 thermostat methods. In addition, the pressure was weakly coupled to a reference pressure of 1.0 bar, a 4.5×10−5 compressibility bar−1 and a coupling constant of 5 ps. The non-bonded Lennard-Jones (LJ) and Coulomb interactions decreased to zero at 12 Å; a shifting function was used from 9 to 12 Å for the former and 0 to 12 Å for the latter. Different temperatures were used in the two current lines of CG simulations considering a good experimental match with the raft-excluded bulk phase 46 and an efficient phase segregation for the formation of lipid rafts 43. The temperature controlled the atom movement but did not affect the secondary structure of the peptide, which was constrained under the Martini condition. In addition, the lower temperature of 295 K did not reach the DPPC crystalline point in the CG model 46, which ensured the comparative DPPC structures in the two bilayer systems. Therefore, it could not produce substantial deviations in the interaction between the peptide and the lipids. The periodic boundary condition was employed, and the integrated timestep was set as 20 fs. The neighbor list of the pairwise non-bonded interactions was updated every 10 steps. Each simulation was performed for an effective time of 4.0 µs. All representations and snapshots were obtained and performed using VMD software 57. Atomistic model and details. The atomistic simulation was performed with the Gromacs-4.6.7 package using the gromos-54A7 force field 58, which was more adaptive for the helical transmembrane peptide. The atomistic simulation was performed for 50 ns, and the initial membrane-peptide structure was obtained from one typical frame of the CG simulation by back-mapping the frame to the defined grained model (containing ~160,000 atoms) by backward scripting 59. The membrane was then solvated by SPC waters 60, and chloridions were added to maintain the system neutral. The system was first energy minimized using the steepest descent method to the ultimate tolerance of 10 kJ·mol-1·nm-1. Subsequently, NVT equilibration was 3



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conducted for 200 ps to separate the coupled groups in the system using the Berendsen thermostat method with a reference temperature of 295 K and a time constant of 0.1 ps. The van der Waals force was decreased to zero at a cutoff distance of 1.2 nm. Particle Mesh Ewald (PME) 61 was applied for long-range electrostatics with a real-space cutoff of 0.9 nm and a 0.12 nm grid spacing. Then, NPT equilibration was performed for 500 ps using the semi-isotropic pressure coupling style. The pressure coupling was implemented using the Parrinello-Rahman method 62 with a reference pressure of 1.0 bar, a compressibility of 4.5×10-5 and a time constant of 2 ps. Note that during the equilibrations, the position of the peptide was constrained by a force of 1000 kJ·mol-1·nm-1 in three-dimensional directions. The periodic boundary condition was applied. The time step of the simulation was set as 2 fs, and the pair list was updated every 10 steps.

Figure 1. Presentations of the peptide and lipids and the segregation of the mixed bilayers. (A) The N-helix domain (688-694) was linked to the transmembrane domain with a short loop. The relevant cleavage residues V711 and A713 were recognized by γ-secretase and are highlighted in green and red, respectively. (B) Representation of the CG models of DPPC, DAPC, DPSM and CHOL. The backbone beads of the lipids included the following: phosphate (yellow), choline (green), glycerol (purple), sphingosine (olive), butenyl group (light brown), saturated alkyl groups (sea blue), unsaturated alkyl groups (red), hydroxyl (brown), and the ring structure (sky blue and orange). The atom types defined in the Martini force field were also stamped. (C) Semi-top views of the initial membrane model (upper) and the phase-segregated membrane at the end of the simulation (bottom) in which DPPC, DAPC, CHOL and DPSM are shown in blue, red, orange and yellow beads, respectively. The slabs of the solvents are shown in light blue.

RESULTS CG model of the raft-forming membranes. To investigate the effect of the membrane microenvironment on the behavioral preference of the APP-TM dimers, we performed a set of molecular dynamic simulations 4


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using the Martini CG model. The Martini force field has been successfully used in studies exploring bilayer segregation 63-64 and interactions among TM helixes 37, 51. To present a realistic raft-forming membrane environment, appropriate amounts of sphingomyelin (DPSM), which act as indispensable raft companions in neural membranes 65-67, were recruited in the bilayer model. Based on existing simulation studies and our trials of the phase-segregated bilayers, a bilayer composed of DPPC/DAPC/CHOL/DPSM at a ratio of 35:20:25:15 was constructed (Figure 1B). After a simulation of 4.0 µs, the lipid mixture segregated successfully into an apparent binary-phase membrane (Figure 1C and S1).

Figure 2. The structural properties of the raft-forming bilayers. (A) Side view of the planar DPPC/DAPC/CHOL/DPSM system at the end of the simulation, and the components are shown in different colors as defined in Figure 1C. The liquid ordered phase was enriched in DPPC and CHOL and is denoted as “Raft”, while the surrounding phase is denoted as “Non-raft”. (B) Thickness evolvements of DAPC (red line) and DPPC (black line) as a function of time. The thickness was calculated according to the distance between the lipid phosphates at the upper leaflet and the lower leaflet. (C) The distribution of the molecular mass density fraction in the final 1.0 µs along the direction perpendicular to the phase boundaries. (D) The average lateral diffusion rates of DPPC, DAPC, DPSM and CHOLs in the final 1.0 µs of the simulations. (E) The order parameters (Sz) of the acyl tails of DPPC and DAPC. For a better comparison, the extra bonds of D4A-C5A and D4B-C5B in DAPC were omitted.

The lipid raft has been compared to the liquid-ordered (Lo) subdomain in bilayer models, and in fact, the Lo phase has been employed as a model raft system in many experiments 23, 34. A lateral snapshot of the segregated bilayer is shown in Figure 2A in which the lipids are partitioned into the rigid raft subdomain and disordered non-raft (liquid-disordered: Ld) region. The organizational distinction between the two domains was elucidated by a thickness differentiation between the saturated/unsaturated lipids, which also indicated that the membrane segregation was accomplished at ~1.5 µs (Figure 2B). To characterize the lateral organization, the density distributions of the components were calculated, and the results demonstrated the divided distributions of DAPC and DPPC (Figure 2B). Meanwhile, CHOL and DPSM mainly localized in the DPPC-enriched domain, which corresponded to the accepted concept in the raft composition. In addition, the DAPC lipids exhibited the highest lateral diffusion rate, which was nearly three-folds greater than that of the DPPC lipids and was consistent with the experimental data 25. Moreover, the more severe disorder of the 5



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non-raft domain was supported by the highly random distribution (Sz closer to 0, implying a random orientation) of the DAPC acyl tails. In contrast, the average order parameter of the DPPC lipids was approximately 0.6 (a value of 1.0 indicates a perfect alignment with the normal bilayer), indicating that the DPPC lipid tails were in good alignment with the normal membrane. Altogether, the mixed membrane model was suitable as a raft-forming bilayer protocol. Table 1. An overview of the behaviors of the C99 variants in different bilayer compositions. Bilayer environments in the simulations

Peptide Variants

Peptide number

Sequences

C99-wt

1

QKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK

C99-1

1

QKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVML

C99-2

1

C99-3

1

C99-wt C99-4 C99-5 C99-6

2 2 2 2

Lipid raft (replicas)

Variable lengths

Non-raft (replicas)

Nhelix

KKK726

√(4)

×

+ + – –

+ – + –

QKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK

√(10)

√(10)

QKLVFFAEDVGSNKGAIIGLMVG IVVI ITVIVITLVMLKKK QKLVFFAEDVGSNKGAIIGLMVG IVVI ITVIVITQVMLIKK QKLVFFAEDVGSNKGAIIGLMVGGGVIATVIGITLVMLKKK

√(4)

×

GAIIGLMVGGVVIATVIVITLVMLKKK GAIIGLMVGGVVIATVIVITLVML

+

Note: The substituted residues are highlighted in bold and red in the sequence lines. Not all peptide variants were subjected to the simulations in both the raft-forming and raft-excluded membranes. The environment condition employed for each peptide variant in this study is denoted by “√”, and the non-employed environment condition is denoted by “×”. The variable lengths with and without the two terminal regions are denoted by “+” and “–”, respectively.

Localization and orientation of C99 in the raft-forming bilayers. First, a bilayer mixture incorporating one C99 monomer was constructed to investigate the molecular details of the peptide-lipid interplay (Table 1). In addition to the phase partition of the bilayer, the C99 monomer was found to localize at the border of the raft subdomain (Figure 3A). This phenomenon was then verified by the 2-dimensional density profile that presented the relative positions of the C99 monomer and the raft component of DPPC (Figure 3B). Moreover, even if the C99 monomer was preplaced in the raft or the no-raft subdomains at the onset of the simulations, consistently in both cases, C99 ultimately wandered to the lipid raft boundary (Figure S2 and S3).

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Figure 3. The preference of the localization and configuration of the C99 monomer in the phase-coexisting bilayers. (A) The top (upper) and lateral (bottom) views of C99 at the boundary of the Lo/Ld subdomains. C99 is represented in green, and the lipid colors are the same as those defined in the scheme described in Figure 1C. (B) The mass density profiles of DPPC and the C99 monomer on the x-y panel. The profile was generated using the script tool 68 by first centering the peptide. (C) The DPPC neighbor contact ratio of the C99 residues. More details are available in SI text. The residue codes of 1-41 represent the C99 residues from the N- to the Cterminal. (D) The DPPC neighbor contact ratios of three C99 variants with different lengths. To clarify the DPPC-binding regions, only the TM domains and KKK residues (if contained) are shown without error bars. All contact data were collected in the final 2.5 µs of all four replicates.

Additionally, the orientation of the C99 TM axis relative to the raft subdomain presented as a lopsided mode, and the C-terminal was embedded into the raft domain, while the N-terminal was located in the nonraft domain (Figure 3A). Based on the different contacts of the peptide with DPPC and DAPC (Figure S4), the DPPC neighbor contact ratios of the C99 residues showed that the C-terminal of VMLKKK726 was buried in the raft with 75-90% of the DPPC contact ratios (Figure 3C). A gradual decrease in the DPPC contacts was observed from the C-terminal to the N-terminal of the C99 TM, particularly at IGLMVG708 (less than 30%). The lopsided orientation of the C99 monomer was further demonstrated by the lateral density distribution, which reflected the predominant affinity of the C99 C-terminal in the lipid raft phase (Figure S5). Note that an intensive DPPC contact was also observed at the short extracellular helix (QKLV688), which possessed a high rotational freedom around the TM helix 16. The lopsided orientation of C99 was likely due to a hydrophobic mismatch of the peptide contacting with the raft and non-raft domains with different thicknesses 69-70. Therefore, peptide variants with sequential truncations of the extracellular domain and the juxta-membrane lysine residues were subjected to the simulations (Table 1). No significant change occurred in the three variants, which showed a common 7



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asymmetric DPPC contact distribution along the N-terminal and C-terminal to the C99-WT (Figure 3D). Therefore, the difference in thickness between the neighboring bilayer domains did not lead to the unbalanced peptide orientation relative to the raft boundary face. However, note that KKK726 in both the WT and C99-2 showed a remarkable contact with DPPC. This contact likely contributed to the increasing electrostatic interactions between the lysine and exposed phosphate groups due to the increased thickness of the raft domain. Moreover, we observed that the raft-affinity residues of the variant were highly conserved in the motif of V710xxA713xxxV717xxxV721, an interface enriched with hydrophobic residues. Based on these results, the discrepant residue capabilities to associate with the lipid components determined the preferential lateral localization and structure of C99 in the raft-forming membrane. The detailed mechanism is analyzed in the following section. Suppressed and transformed C99 dimerization. To determine the effects of the membrane composition on the dimerization of the C99s, two monomers were incorporated into the mixed membrane and were separated from each other by 55 Å. Meanwhile, a pure DPPC bilayer coupled at a higher temperature 71-73 was employed to imitate the bulk raft-excluded environment to eliminate the influence of the lipid raft. Compared with the rapid and successful dimerization of all 10 replicates in the bulk bilayer environment, 7 samples in the raft-forming bilayers could self-associate into homodimers at different rates, and 3 samples required nearly 1.5 µs to accomplish the dimerization. Meanwhile, two other samples failed to pack together throughout the simulations, and a disassociation event occurred in the last replicate (Figure 4A). In addition, it should be noted that the C99 peptides, even in the suppressed dimerization, remained at the border of the raft subdomain, which is consistent with the occurrence of monomerization (Figure S6).

Figure 4. Comparisons of the dimerization and conformation of C99s in the raft-forming bilayers (A, B, and C) and the raft-excluded bilayers (D, E and F). (A) and (D) The distance evolvements between the backbones of two C99 monomers as a function of time. Ten independent runs were considered, respectively. (B) and (E) The dihedral angle distribution of the C99 dimers. The data were collected from all trajectories in which the C99 dimerization was maintained. The inserted figures represent the respective chiral dimer modes. G709, V711 and A713 are represented as yellow, gray and purple beads, respectively. The backbones of the peptide chains 8


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are shown in green and red. (C) and (F) The residue contact matrixes of the predominant left-handed dimers formed in the raftforming membranes and the right-handed dimers in the raft-excluded membranes.

Furthermore, the dimer structure was unstable under the influence of the lipid rafts. The dimer structure showed a bimodal dihedral angle distribution that varied from the 20˚ left-handed to -10˚ right-handed confirmations alternatively (Figure 4B), which implied the switched dimeric configurations of C99 in the presence of the lipid raft. Regarding dimerization in the bulk fluid membranes, the peptide package exhibited a stable -20˚ right-handed conformation (Figure 4E) with a G709xxxA713 motif on the interface (Figure 4E and F). In contrast, in the raft-forming environment, the left-handed configuration became predominant. Meanwhile, the residues V711 and V715 began to occupy the interface (Figure 4B, 4C and S7). Molecular mechanism. As a crucial component in the raft inclusion, the role of CHOL in mediating APP dimerization and generating Aβ peptides has been extensively studied 74. Unlike previous modeling studies using a single membrane phase 9, 17-18, CHOL in a phase-coexisting bilayer is restrictedly available in lipid rafts. Calculating the contact intensity between C99 and CHOL in the peptide dimerization state showed that the predominant CHOL contacts occurred at the motifs G709xxxA713 and L720xxxK724, which were distributed throughout the C-terminal (Figure 5A). The time-averaged spatial distribution of CHOL around C99 also showed the priority of the tail domain bound by CHOL (Figure 5B). This phenomenon was further verified by the asymmetrical radial distributions of CHOL in the two half TM segments around which the enrichment of CHOL in the C-terminal was quite apparent (Figure 5C). To reveal the molecular details, the mutant G709IA713I (C99-4) was simulated to monitor changes in the CHOL attachment on C99. Substitutes of the isoleucine residues were used to increase the bulk and hydrophobic level of the region. The C99-4 mutation resulted in an apparent decline in the CHOL contacts at the G709xxxA713 region in the WT, but the enrichment of CHOL in the L720xxxK724 region persisted. If all CHOL binding sites were removed at G709IA713IL720QK724I (C99-5), the mutation could basically eliminate the prominent CHOL contact regions, leading to a moderate contact distribution along the C99 TM region (Figure 5A). These results indicated that the G709xxxA713 motif interacted with CHOL by specific van de Waals interactions, while CHOL-OH associated with K724 by forming hydrogen bonds. These results inferred that CHOL mostly adopted its smooth α-surface to allow the formation of CH2- π stacking between the sterane rings and apolar L720, allowing the β-surface occupied with methyl groups to reside in the lipid raft enriched with saturated carbon bonds. Considering that CHOL can flip-flop to a certain extent 75 and associate with residues in numerous ways 40, 76, the CHOL-C99 contact interface likely resulted from more than one interaction mode.

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Figure 5. (A) The CHOL contact distribution along the C99 TM region in the dimerization of the WT, C99-4 and C99-5. The data were collected from four replicates of the WT and mutants as they formed stable dimers after 1.5 µs. (B) The spatial isosurface distribution of the attached CHOL around the peptide in dimerization. The spatial density distribution was plotted and averaged from data obtained during the final 1.0 µs of the simulation. The CHOL and peptide are shown in red and green, respectively. More details are shown in SI text. (C) The radial distribution functions of CHOL in the upper and lower bilayer leaflets in the C99 TM N-terminal and C-terminal segments, respectively. (D) The initial and final snapshots of the atomistic simulation. Lipid representation is consistent with the upper description. C99 was rendered depending on the secondary structures. V710, V717, V721 are showed by the “VDW” style in orange, and A713 is shown in green. (E) Root mean square deviation (RMSD) of C99 as a function of the simulation time.

As discussed above, the C99 monomer interacted with the DPPC lipids by the conserved V710xxA713xxxV717xxxV721 motif. By transferring the CG model (state of Figure 3A) to an atomistic structure and simulating this model for 50 ns, the orientation of the quaternary motif showed good conservation as it rotated slightly relative to the raft domain (the top and bottom views shown in Figure S8), except for the upmovement of the short extracellular helix. C99 was considerably stable by the early flat trend of peptide RMSD at approximately 0.37 nm. To determine whether the structural characteristics remain under the condition of dimerization, the DPPC neighboring contact ratios for the dimers of C99-WT, C99-4 and C99-5 were calculated (Figure 6A). The result demonstrated that the C99 dimer adopted the V710xxA713T714xxV717xxxV721 motif to interact with DPPC lipids, which was highly conserved with the monomeric case. Furthermore, replacing V710 and V717 with small polar glycine residues (V710GV717G, C99-6) manifested alterations in the raft-contact mode, and the DPPC-affinity interface converted to the region of V711xxxV715xxI718 (Figure 6B). Meanwhile, the residue substitutes G710 and G717 showed a sharply reduced DPPC-contact, indicating a great deviation from the lipid raft. The orientation transformation was derived from the different affinities of the residues to the saturated and unsaturated lipids. Since acyl double-bonds 10


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with highly polarizable π-electrons exhibit a higher hydration than saturated bonds 38, 77-78, the unsaturated DAPC became more polar than the saturated DPPC. This property discrepancy, therefore, enabled the small polar glycine to preferentially associate with the unsaturated DAPC lipids, while the hydrophobic residues, such as valines, matched the DPPC lipids due to the favorable hydrophobic interaction. Similar to that of C99-5, in which all basic CHOL contact sites were abrogated, the lateral orientation of the C99-6 TM domain became more balanced in the contact to the raft boundary face. Based on these results, the lateral localization and orientation of the APP TMs were maintained by the combined regulation of CHOL and saturated lipids in the raft-forming bilayers. The distribution of the residues with a high affinity to the lipid raft was intermittent along the C99 TM, and the involved residues that could modify the orientation specificity were removed. The structure switching is potentially associated with the directional biological expression. As shown in Fig. 6B, the apparent positional switch of the amyloidogenic sites V711 and A713 relative to the raft domain could result from the C99-6 mutation. To more clearly demonstrate this phenomenon, the DPPC neighbor ratios between the WT and C99-6 were compared (Figure 6C). The neighboring DPPC ratio of A713 in the WT declined apparently compared with that in the mutant. In contrast, the ratio of V711 increased significantly from 20% (WT) to 50% (C99-6). The switched DPPC affinity corresponded to the reverse residue arrangement relative to the lipid raft (Figure 6D). Accordingly, the results indicated the selectivity of the lipid raft in binding the TM residues of C99, which restricted the structure and orientation of the TM region present in the membrane.

Figure 6. (A) The DPPC neighboring ratio of the individual residues on the C99-WT dimers and the two mutants. The residues V711 and A713 are marked by different arrows to highlight their discrepant affinities to the lipid rafts. The dominant DPPC contact residues are noted. (B) The DPPC neighbor ratios in C99-WT and C99-6. The mutant 710th and 717th sites are shown by the blue arrows. For clarify, the TM regions are shown without the final VML723. (C) A comparison of the DPPC contact ratios of A713 and V711 between the WT and C99-6. (D) Presentation of the positional alteration in the V710xxA713T714xxV717xxxV721 motif relative to the lipid raft in 11



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the WT and C99-6. The backbone atoms of the valine, threonine and substituted glycine residues are, respectively, represented as orange, yellow and purple beads. The DPPC lipids are shown in the silver bulk block. Note that the snapshots are shown in the bottom view.

DISCUSSION The influence of special inclusions in membranes and phase segregation is considered increasingly important in protein translocation and linkage that control biological processes, such as peptide clustering 42 and cell migration 79. Compartmentalized bilayers are believed to play a critical role in peptide sorting and conformation, particularly in the GPI- and palmitoyl-anchored proteins 80. Lipids regulate the trafficking/proteolytic activity of APP and its intimate enzyme β-secretase β-site APP cleavage enzyme 1 (BACE1), which plays a fundamental role in AD. Studies investigating the unilamellar vesicles have shown the CHOL-dependent activity of γ-secretase, which links the interrelation between a CHOL-enriched lipid raft and amyloidogenic processing of APP. In this study, we used time-extended Martini CG simulations to obtain insights regarding the structural deformation details of (dimeric) C99 between different bilayer compartments. In the bulk fluid phase, C99s form stable right-handed homodimers that interact with the G709xxxA713 motif, which is consistent with the NMR data 14. However, the motif crosslinking is abolished when C99 dimerizes near the CHOL-enriched raft, where CHOL competitively binds to G709xxxA713. These results are consistent with existing data showing that CHOL is unfavorable for C99 dimerization 8, 16, 81. Moreover, the dimerization is unstable, and switching occurs to a large extent under the influence of the lipid raft. The right-handed dimeric mode, which is packed by the G700xxxG704xxxG708 motif, appears in this situation but presents as a subsidiary mode (Figure S9). In fact, a recent study using complicated bilayers also shows that the sensitive alterations in the binding interfaces vary with the bilayer components 12, 18. Epidemiological evidence suggests that an elevated CHOL level increases the risk of developing AD82, potentially suggesting that there is a connection between the CHOL-enriched phase and the structure preference of APP. In this study, although the dimerization ability and conformation are largely affected by the lipid raft, the orientation of the C99 TM domain in the raft is highly conserved at the V710xxA713T714xxV717xxxV721 motif (Figure 5D). However, C99 monomers and homodimers were not observed to obviously enter the lipid raft in this study. Considering previous work on the partition of the transmembrane WALP, which always remains in the non-raft phase but merely translocates to the raft boundary by introducing as many as eight palmitoyls 83, we believe that C99 resides at the raft boundary due to the guiding of the lipid raft. Note that the raft-association motif is mostly hydrophobic, except for T714. The driving force is likely attributed to the additive interaction derived from CHOL because CHOL is positionally active at the boundary of the raft. This deduction is supported by the higher DPPC contact of G709 than V711, reflecting the flexible and versatile interaction modes of CHOL that contains polar and hydrophobic groups 40, 84. The combined regulation of the raft components finally enables the C99 dimeric structure to transform from the -20° right-handed mode to the dominant 20 left-handed packing mode, even if the dimerization is detached. One explanation of this phenomenon is the weak binding of V711xxxV715 in the new left-handed homodimer. The -10° right-handed dimeric mode packed by the G700xxxG704xxxG708 motif presents a secondary structure under the influence of the lipid raft. Combined with the asymmetrical raft-contact of the CD99 TM domain, this phenomenon is mainly caused by the intensive unsaturated lipid interaction in the N-terminal region of C99 TM because of the affinity of unsaturated lipids to C99 glycineenriched N-terminal 78. This mechanism also explains the evident alteration in C99-6 in contacting DPPC, which contributes to the enhanced association of the substituted polar glycine to DAPC in the non-raft subdomain (Figure 5E). The sensitivity of the APP dimeric structure is impacted by the membrane 12


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composition, and it has been reported that C99 homodimers display interconverted packing states with the Nterminal glycine zipper in or out of the dimer interface as a function of the lipid compositions 85. Therefore, it is not surprising that the N-terminal G700xxxG704xxxG708 region is mostly situated in the non-raft subdomain (Figure 3C). Due to the conserved orientation of the C-terminal facing the lipid raft with G709xxxA713 on the C99 dimeric interface, the package formation of the G700xxxG704xxxG708 motif becomes unfavorable.

Figure 7. Presentation of the transformation of C99 dimerization, structure and proposed amyloidogenic processing. C99 dimerization shows a stable -20˚ right-handed mode in the bulk non-raft phase, exposing V711. Due to the reduced availability of γ-secretase and the concealment of A713 under this condition, the cytotoxic amyloid forms generate in an inactive state. When C99 associates with the lipid raft, the dimer begins to transform to the left-handed mode or dissociate from the monomers. The resulting peptide orientation is favorable for A713, which is recognized by γ-secretase that prefers to associate with the raft domain. In this case, the generation of the amyloid forms is accelerated, particularly Aβ42. The amount and proportion of the amyloid substrates and bilayer phase backgrounds are depicted. The thunder symbols with different sizes indicate the capability of γ-secretase. The bottom membrane models represent the bulk non-raft (left) and raft-forming (right) bilayers.

It has been reported in previous in vitro assays that the mutant V717G affects the generation of Aβ species . Thus, the raft-associated residues greatly restrict the lateral rotation of the APP TM. Consistently with the experiment, the substitution of V710xxxV717 in the simulation leads to a remarkable switch of the DPPCassociation residue array in which V711 (amyloidogenic site of Aβ40) converts to directly contact the raft domain. This phenomenon is related to the evidence that γ-secretase has a higher ability to associate with lipid rafts to modulate the peptide trafficking and structure 27, 86. Previous studies using artificial lipid vesicles and simulations further proposed that CHOL-rich lipid rafts regulate the amyloidogenit process based on CHOL’s inhibition of C99 dimerization 8, 16. Based on the estimations of raft-associated sphingolipid in the membrane, the area of the raft compartments could cover more than half of the cell surface theoretically 81. Therefore, it is more realistic to investigate the C99 dimerization in raft-forming bilayers. Moreover, increasing evidence shows that only the monomeric APP can be cleaved by γ-secretase in the released amyloid forms 14, 87-89. This phenomenon, thus, is consistent with the depressed dimerization of C99, which is influenced by the lipid raft with which β- and γ-secretase are prone to associate as supported by numerous reports 90-91. Moreover, it is energy favorable for the C99 in monomerization to enter 14

13



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the rigid lipid raft. In addition, we also observed a high raft affinity in the short ectoplasmic domain of C99 in its monomeric condition (Figure 3C). The cleavage of α-secretase in the extracellular domain yields nonamyloidogenic products, which has been shown to occur outside of the lipid raft 92-93. Thus, it the configuration of C99 induced by the raft subdomain is favorable for the amyloidogenic process. The location preference of the protein in particular membrane subdomains acts as a necessary step for protein sorting and biology pathway switching 31, 94. Previous studies indicated that enhancing the association between APP and the lipid rafts is favorable for amyloidogenic processing 29, 39, 95. Reports have also shown that CHOL interacts with the C-terminal residues 22-40 of Aβ40, while the contact distribution shifts to the N-terminal residues of 22-28 of full-length APP 16-17, 96. In this study, CHOL-association mainly concentrated at the C-terminal region of the C99 TM domain; thus, the destabilized structure induced by the lipid raft is readier for the processing of Aβ release. The APP TM domain contains the key proteolytic sites of A713 and V711, which, respectively, produce Aβ42 and Aβ40 after cleavage by γ-secretase. As shown in Figure 7, when the dimerization of C99 is suppressed by lipid rafts, C99 in monomer or homodimer consistently adopts the V710xxA713T714xxV717xxxV721 motif to face the raft domain. γ-secretase, therefore, has more accessibility to the A713 cleavage site to generate more cytotoxic Aβ42. In comparison, the site V711, which is associated with Aβ40, is protected from proteolysis in the dimer interface or on the raft-opposite side (Figure 4C and 6C). The structure conservation, thus, provides a referential interpretation of raft promoting the amyloidogenic process. Previous studies have shown that there is an elevated Aβ42/Aβ40 ratio in many cases of familial AD 4, 14 , and in fact, a slight increase in Aβ42 can lead to a severe neurological disorder 7. Therefore, lipid rafts play a key role in mediating the amyloidogenic pathway to accelerate fibrillogenesis processing. It has been proposed that the cleavage specificity at the Aβ42 and Aβ40 residues of APP is sensitive to the bilayer thickness 27. The evidence provides direct molecular clues regarding the regulation role of lipid rafts in Aβ generation. Furthermore, lipid rafts can promote the fibrillogenesis of soluble Aβ, suggesting that the raft association can result in a conformational change favoring the formation of amyloid plaques 97. Notably, the cleavage of APP can be affected by more than one factor, such as the CHOL concentration and activity level of γ-secretase. Reports show that gangliosides, such as GM1, can bind Aβ peptides and induce them to form more ordered structures with increased cytotoxicity 98. More explorations are required to outline the adjustable amyloidogenic pathways that rely on advanced membrane environments, structural deformation in protein associations and longer time-scale perspectives for amyloidogenic processing.

CONCLUSIONS In this study, we used CG dynamic simulations to explore the dimerization, localization preference and structure deformation of APP transmembrane substrates in molecular details. C99 preferred to reside at the boundary of the raft subdomain in monomers and homodimers. Moreover, C99 associated with the lipid raft via the conserved V710xxA713xxxV717xxxV721 motif. Due to the combined regulation of CHOL and saturated lipids, dimerization of the C99 TM domains was inhibited and showed a susceptible conformation compared with that of the stable dimers in the bulk non-raft phase. The resulting dimerization and structure were likely involved in the downstream amyloidogenic alternations. Our study provides molecular insights regarding the role of CHOL-enriched compartments in mediating peptide self-association and our interpretation of the structural requisites for releasing cytotoxic entities.

Supporting information Information regarding the methods and analysis and the supporting materials are available online at 14


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http://pubs.acs.org/

* Corresponding authors: [email protected]; [email protected] Notes The authors declare no competing financial interests.

Acknowledgments This work was supported by the National Basic Research Program of China (973 program) (2013CB910700), the National Natural Science Foundation of China (21372026, 21402006, 61304147 and 21672019), the Fundamental Research Funds for the Central Universities (Grant No. XK1701, YS1407 and 2050205), BUCT Fund for Disciplines Construction (Project No. XK1701), and the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (The second phase). We also thank Ph.D. Floris van Eerden to help improve the article.

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Figure 1. Presentations of the peptide and lipids and the segregation of the mixed bilayers. (A) The N-helix domain (688-694) was linked to the transmembrane domain with a short loop. The relevant cleavage residues V711 and A713 were recognized by γ-secretase and are highlighted in green and red, respectively. (B) Representation of the CG models of DPPC, DAPC, DPSM and CHOL. The backbone beads of the lipids included the following: phosphate (yellow), choline (green), glycerol (purple), sphingosine (olive), butenyl group (light brown), saturated alkyl groups (sea blue), unsaturated alkyl groups (red), hydroxyl (brown), and the ring structure (sky blue and orange). The atom types defined in the Martini force field were also stamped. (C) Semi-top views of the initial membrane model (upper) and the phase-segregated membrane at the end of the simulation (bottom) in which DPPC, DAPC, CHOL and DPSM are shown in blue, red, orange and yellow beads, respectively. The slabs of the solvents are shown in light blue. 352x267mm (300 x 300 DPI)



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Figure 2. The structural properties of the raft-forming bilayers. (A) Side view of the planar DPPC/DAPC/CHOL/DPSM system at the end of the simulation, and the components are shown in different colors as defined in Figure 1C. The liquid ordered phase was enriched in DPPC and CHOL and is denoted as “Raft”, while the surrounding phase is denoted as “Non-raft”. (B) Thickness evolvements of DAPC (red line) and DPPC (black line) as a function of time. The thickness was calculated according to the distance between the lipid phosphates at the upper leaflet and the lower leaflet. (C) The distribution of the molecular mass density fraction in the final 1.0 µs along the direction perpendicular to the phase boundaries. (D) The average lateral diffusion rates of DPPC, DAPC, DPSM and CHOLs in the final 1.0 µs of the simulations. (E) The order parameters (Sz) of the acyl tails of DPPC and DAPC. For a better comparison, the extra bonds of D4A-C5A and D4B-C5B in DAPC were omitted. 344x186mm (300 x 300 DPI)


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Figure 3. The preference of the localization and configuration of the C99 monomer in the phase-coexisting bilayers. (A) The top (upper) and lateral (bottom) views of C99 at the boundary of the Lo/Ld subdomains. C99 is represented in green, and the lipid colors are the same as those defined in the scheme described in Figure 1C. (B) The mass density profiles of DPPC and the C99 monomer on the x-y panel. The profile was generated using the script tool 68 by first centering the peptide. (C) The DPPC neighbor contact ratio of the C99 residues. More details are available in SI text. The residue codes of 1-41 represent the C99 residues from the N- to the C-terminal. (D) The DPPC neighbor contact ratios of three C99 variants with different lengths. To clarify the DPPC-binding regions, only the TM domains and KKK residues (if contained) are shown without error bars. All contact data were collected in the final 2.5 µs of all four replicates. 281x228mm (300 x 300 DPI)



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Figure 4. Comparisons of the dimerization and conformation of C99s in the raft-forming bilayers (A, B, and C) and the raft-excluded bilayers (D, E and F). (A) and (D) The distance evolvements between the backbones of two C99 monomers as a function of time. Ten independent runs were considered, respectively. (B) and (E) The dihedral angle distribution of the C99 dimers. The data were collected from all trajectories in which the C99 dimerization was maintained. The inserted figures represent the respective chiral dimer modes. G709, V711 and A713 are represented as yellow, gray and purple beads, respectively. The backbones of the peptide chains are shown in green and red. (C) and (F) The residue contact matrixes of the predominant left-handed dimers formed in the raft-forming membranes and the right-handed dimers in the raft-excluded membranes. 339x200mm (300 x 300 DPI)


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Figure 5. (A) The CHOL contact distribution along the C99 TM region in the dimerization of the WT, C99-4 and C99-5. The data were collected from four replicates of the WT and mutants as they formed stable dimers after 1.5 µs. (B) The spatial isosurface distribution of the attached CHOL around the peptide in dimerization. The spatial density distribution was plotted and averaged from data obtained during the final 1.0 µs of the simulation. The CHOL and peptide are shown in red and green, respectively. More details are shown in SI text. (C) The radial distribution functions of CHOL in the upper and lower bilayer leaflets in the C99 TM N-terminal and C-terminal segments, respectively. (D) The initial and final snapshots of the atomistic simulation. Lipid representation is consistent with the upper description. C99 was rendered depending on the secondary structures. V710, V717, V721 are showed by the “VDW” style in orange, and A713 is shown in green. (E) Root mean square deviation (RMSD) of C99 as a function of the simulation time. 325x215mm (300 x 300 DPI)



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Figure 6. (A) The DPPC neighboring ratio of the individual residues on the C99-WT dimers and the two mutants. The residues V711 and A713 are marked by different arrows to highlight their discrepant affinities to the lipid rafts. The dominant DPPC contact residues are noted. (B) The DPPC neighbor ratios in C99-WT and C99-6. The mutant 710th and 717th sites are shown by the blue arrows. For clarify, the TM regions are shown without the final VML723. (C) A comparison of the DPPC contact ratios of A713 and V711 between the WT and C99-6. (D) Presentation of the positional alteration in the V710xxA713T714xxV717xxxV721 motif relative to the lipid raft in the WT and C99-6. The backbone atoms of the valine, threonine and substituted glycine residues are, respectively, represented as orange, yellow and purple beads. The DPPC lipids are shown in the silver bulk block. Note that the snapshots are shown in the bottom view. 300x212mm (300 x 300 DPI)


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Figure 7. Presentation of the transformation of C99 dimerization, structure and proposed amyloidogenic processing. C99 dimerization shows a stable -20˚ right-handed mode in the bulk non-raft phase, exposing V711. Due to the reduced availability of γ-secretase and the concealment of A713 under this condition, the cytotoxic amyloid forms generate in an inactive state. When C99 associates with the lipid raft, the dimer begins to transform to the left-handed mode or dissociate from the monomers. The resulting peptide orientation is favorable for A713, which is recognized by γ-secretase that prefers to associate with the raft domain. In this case, the generation of the amyloid forms is accelerated, particularly Aβ42. The amount and proportion of the amyloid substrates and bilayer phase backgrounds are depicted. The thunder symbols with different sizes indicate the capability of γ-secretase. The bottom membrane models represent the bulk nonraft (left) and raft-forming (right) bilayers. 323x177mm (300 x 300 DPI)



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TOC Graphic 288x164mm (300 x 300 DPI)


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Dimerization and Structural Stability of Amyloid Precursor Proteins

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