1 Mechanism of Initiation, Association and Formation of Amyloid Fibrils

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Mechanism of Initiation, Association and Formation of Amyloid Fibrils Modeled with the N-terminal Peptide Fragment, IKYLEFIS, of Myoglobin G-Helix Sunita Patel, Yellamraju U. Sasidhar, and Kandala V.R. Chary J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b02205 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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The Journal of Physical Chemistry

Mechanism of Initiation, Association and Formation of Amyloid Fibrils Modeled with the N-terminal Peptide Fragment, IKYLEFIS, of Myoglobin G-helix

Sunita Patel1,2*, Yellamraju U. Sasidhar3, Kandala V. R. Chary1,4 1

2

Tata Institute of Fundamental Research, Center for Interdisciplinary Sciences, Hyderabad, India

UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Mumbai, India 3

Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, India 4

Tata Institute of Fundamental Research, Mumbai, India

*Corresponding address: Dr. Sunita Patel Tata Institute of Fundamental Research, Centre for Interdisciplinary Sciences, Hyderabad, 500075, INDIA Current address: UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Mumbai 400098, India Email: [email protected] Tel.: 91-22-26524983 Fax: 91-22-26524982

1 ACS Paragon Plus Environment


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ABSTRACT Some peptides and proteins undergo self-aggregation under certain conditions, leading to amyloid fibrils formation, which is related to many disease conditions. It is important to understand such amyloid fibrils formation to provide mechanistic detail that governs the process. A predominantly α-helical myoglobin has been reported recently to readily form amyloid fibrils at a higher temperature, similar to its G-helix segment. Here, we have investigated the mechanism of amyloid fibrils formation by performing multiple long molecular dynamics simulations (27 µs) on the N-terminal segment of the G-helix of myoglobin. These simulations resulted in the formation of a single-layered tetrameric β-sheet with mixed parallel and antiparallel β-strands and this is the most common event irrespective of many different starting structures. Formation of the single-layered tetrameric β-sheet takes place following three distinctive pathways. The process of fibril initiation is dependent on temperature. Further, this study provides mechanistic insights into the formation of multi-layered fibrilar structure, which could be applicable to a wider variety of peptides or proteins to understand the amyloidogenesis.


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INTRODUCTION Polypeptide chains coming out of ribosomes follow two folding pathways depending upon the cellular conditions1. Most of the time, they follow a pathway that involves spontaneous search of a conformation accessible to them. In such a folding process, the near-native interactions having lower free-energy are favored over far-native interactions2. Such folding process results in a definite three-dimensional (3D) structure for the protein. On the other hand, some proteins misfold or/and aggregate leading to the formation of amyloid fibrils, which could trigger diseases such as Alzheimer's disease, type II diabetes, Huntington’s disease, Parkinson’s disease etc.

2-6

.

Recently, many globular proteins, which are not associated with any diseases, were also shown to form amyloid fibrils under certain condition(s). For example, low pH7, high temperature8, high pressure9, the presence of co-solvents10 or a point mutation11 could promote partial unfolding of the protein, which in turn may result in amyloid fibrils. The amyloid fibril formation has three distinct phases: nucleation, growth and saturation12-13. The nucleation phase is the most crucial step of amyloidogenesis12,

14

. This process is thermodynamically unfavorable therefore it

becomes a rate-limiting step. Once such nucleus forms, further growth into amyloid fibril is spontaneous and faster. The experimental characterization of the initial stages of such amyloid formation is non-trivial, because of the transient nature of the intermediate conformations. Therefore, investigation of such a recondite area is crucial to understand the amyloid initiation, association and formation, which could then help us in the development of strategies to overcome the amyloid formation and reverse the process. In 1969, X-ray fibril-diffraction showed for the first time that amyloid fibrils adopt an ordered cross β-structure15-16, in which individual β-sheets were arranged parallel to the fibril axis, while monomer β-strand units were perpendicular to the fibril axis. The 3D structure



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determination of such amyloid fibrils in atomic detail is challenging due to the non-crystalline (amorphous) and insoluble nature of the amyloid fibrils that prevents the use of both highresolution X-ray crystallography and solution-state NMR spectroscopy for their structural studies. Instead, solid-state NMR techniques or molecular dynamics (MD) simulations are found to be useful alternate methods17-19. Recently, MD20, replica exchange molecular dynamics (REMD)

21

, coarse-grained simulations19, 22 and several other computational and experimental

studies12, 23-25 addressed various amyloid related queries. A majority of these studies concentrated on the stability and dynamics of small oligomers14,

26-30

, while very few studies explained

mechanistic view underlying the fibril formation, which is important for an in-depth understanding of amyloidogenesis. More recently, it has been established that virtually any protein under certain condition(s) could form amyloid fibrils, irrespective of the information encoded in the primary structure1, 4, 6. Thus, amyloid formation is assumed to be a generic property of any given polypeptide chain. Interestingly, several globular proteins having α-helical structure in their native state were also shown to form amyloid fibrils31-32. Apo-myoglobin (heme-free myoglobin), a small α-helical protein found in the muscle, is one among them33. This protein, though not associated with any disease condition as yet, was reported to form amyloid fibrils in-vitro having a cross β-structure34 at a higher temperature (338 K), or in the presence of a denaturant or a point mutation2, 4, 8, 35. Myoglobin protein is responsible for oxygen supply to the muscle tissue in vertebrate mammals. Formation of such amyloid fibrils thus could impair oxygen supply to the muscle and might cause muscle related diseases. A prerequisite for such amyloid fibril formation is the conversion of an α-helical secondary structural element into a β-strand or a β-hairpin16,

35

. For example,

based on the electron micrograph images of the amyloid fibrils, Fandrich et al.35 showed the


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formation of amyloid fibrils with an isolated full-length G-helix of myoglobin (CH3COIKYLEFISQAIIHVLHSR-NH2), at 333 K and pH 5. However, the mechanism governing such fibril formation was not discussed. In another study, based on an in-silico analysis, Yoon et al. 36 predicted that even a shorter N-terminal fragment of the G-helix of myoglobin ‘IKYLEFIS’ possess a hidden propensity for amyloid fibril formation. However, a generalized mechanism behind such amyloid fibril formation still remained unclear. In this paper, we investigated the mechanism of amyloid fibril formation in a step-by-step process by performing multiple long MD simulations on Ac-IKYLEFIS-NMe peptide in explicit water. All the simulations performed totaled to 27 µs, with each individual simulation running for 1 µs or more. A wide variety of starting structures were used to avoid any conformational bias. This is one among the few thoroughly studied systems to provide complete atomistic details of amyloid fibril initiation, association and formation. Our study provides new insights into the fibril initiation process which shows influence of temperature with mechanistic details. We find that the overall mechanism of amyloid fibril nucleation follows a few common mechanisms. Intriguingly, during this process, we found the formation of a single layered tetrameric β−sheet as the most common event irrespective of many different kinds of starting structures. Further, this study provided a rationale for the formation of higher ordered multi-layered fibrilar structures. Thus, the study provides a detailed mechanism of amyloid fibril formation, which could be applicable to wide variety of peptides and proteins to understand the process of amyloidogenesis.

METHODS Starting structures



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The MD simulation involving the N-terminal eight residues (IKYLEFIS) of the G-helix of myoglobin (Figure 1) was carried out with ten distinct starting structures which were used to perform thirteen different simulations with some simulations repeated twice or thrice with different initial velocity (Table S1). The N- and C-termini of the peptides were capped with Ac(CH3CO-) and -NMe (-NHCH3) groups, respectively for all the simulations, which is done routinely for short peptides to represent the sequence as a part of protein. The starting structures were decided based on the outcome of monomeric peptide simulations performed at 300 and 333 K starting from the native α-helical conformation (PDB id: 1npf)37 which resulted in predominantly extended and β-hairpin conformations. The completely extended structure, where all ϕ and ψ angles are set at 180˚, is used instead of the extended conformation that resulted from the monomer simulation. Such an extended structure is used in literature to study amyloid fibril formation38. The notation to label various simulations thus performed is indicated in Figure 1. The fully extended structure was replicated and translated in the desired direction using Pymol39 to make oligomeric starting structures. The inter-layer distance was set to 10 Å while the intralayer inter-strand distance was set to 5 Å, which is in conformity with the observed crystallographic diffraction pattern for apo-myoglobin amyloid fibrils and other fibrils patterns 16, 35

. All oligomers were prepared in this way except in the case of 4SEPa where all the four

strands were set at 15 Å apart as shown in Figure 1, to minimize inter-strand interactions in the starting structure for this simulation. For 4DEA simulation, all the strands were anti-parallel with respect to each other. For 8DEAm and

16

FEAm simulations, the starting structures were built

from the structure that resulted from 4DEP 333 K simulation by stacking two and four layers of the tetrameric β-sheet respectively such that each β-strand of a given layer is anti-parallel with respect to other β-strand of opposite layer as shown in Figure 1.


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Simulation details All the MD simulations were performed using the software GROMACS (version 5.0.4)40 with explicit solvent. We used the recently re-parameterized united-atom force-field, GROMOS96 54a7 41-42. The electrostatic interactions were treated with Particle-Mesh-Ewald (PME) method4344

with a Coulomb cutoff of 1 nm for monomer and 1.4 nm for oligomers, Fourier spacing of

0.12 nm and an interpolation order of 4. The van der Waals interactions were treated using Lennard-Jones potential with 1 nm cut-off for monomer and 1.4 nm for oligmer simulations. The details of the simulations and analyses are given in the Supplementary Information (SI).

Data analyses Analyses were done on the simulations by measuring various conformational parameters, such as backbone radius of gyration (Rg), root mean square deviation (RMSD) of Cα atoms with respect to end structure of the trajectory following Barz et al.25. Further we determined nematic order parameter (P2), and clustered conformations with network layout (see SI for further details). The strands in a tetramer were referred as C1 (chain 1), C2 (chain 2), C3 (chain 3) and C4 (chain 4). The orientation of the strands in an oligomer is indicated by a superscript (Ci) or subscript (Ci) of the chain identifier, when it is in parallel or anti-parallel orientation, respectively.

RESULTS The monomeric peptide sampled β-hairpin and extended conformations Myoglobin is an α-helical globular protein33 consisting of eight helices, of which G-helix is known to show β-strand propensity and folds early during the protein folding45-46. The G-helix



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was shown to form amyloid fibrils readily at 60 oC in sodium phosphate buffer (pH 3) or sodium acetate buffer (pH 5)35. An in-silico study predicted the hidden amyloid fibril propensity for the shorter N-terminal fragment of myoglobin G-helix, (IKYLEFIS)36. In order to understand the conformational propensities of this fragment, we performed MD simulations on it in explicit water at 300 and 333 K. The higher temperature (333 K) is considered because it is known to promote amyloid fibril formation35. Intriguingly, both at 300 and 333 K, we observed lower and higher values of Rg and RMSD during the entire course of simulations (see Figure S1). The Rg distributions, peak at ~4.7 and ~8 Å suggesting sampling of compact and extended conformations, respectively. The compact and extended conformations sample β-hairpin and coil conformations respectively as shown in the DSSP plot (Figure S1). The network layouts revealed the existence of the abovementioned two distinct conformational forms, extended and β-hairpin, for 79% and 23% of the time at 300 K and for 67% and 33% of the time at 333 K, respectively, suggesting a decrease in extended conformational form and corresponding increase in β-hairpin conformation with increase in temperature (Figure 2). Similar population percentages were obtained by taking Rg cutoff of 5.5 Å to segregate β-hairpin conformations from the extended conformations (see Figure S1 and Figure 2). The apo-myoglobin protein and the G-helix peptide were shown independently to form amyloid fibrils and were studied by electron microscopy, X-ray diffraction, CD, Fluorescence and Fourier-transform IR spectroscopy8,

35

. In the absence of tertiary interactions and under

denaturing conditions, the local interactions dominate and contribute to the overall conformational preferences of any given peptide47. The extended and β-hairpin conformations at 300 and 333 K were manifestation of these local sequence propensities. Therefore, it can be presumed that under a denaturing conditions or in the absence of other tertiary interactions, the


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above-discussed conformations could initiate amyloid fibril formation. The extended and βhairpin conformations were previously shown to mediate aggregation in amyloid β and Prion peptides38,

48-49

. Therefore, the local sequence propensity overrides the global structural

propensity in partially unfolded state(s), which could then serve as an initial event in amyloid fibril formation.

Formation of a stable single-layered tetrameric β-sheet at 333 K The initial event of amyloidogenesis involves formation of a stable nucleus or a seed which usually consists of three to four β-strands arranged in parallel and/or anti-parallel orientations14, 27-28

. Primarily, they are stabilized by inter-strand hydrogen bonding. To understand the influence

of temperature on the formation of such a seed, MD simulations were carried out at three different temperatures (300, 333 and 353 K) with 4DEP as the starting structure (Figure 1). A prior experimental study on apo-myoglobin was shown to form amyloid fibrils at 65 ◦C and any temperature above and below this prohibits fibril formation35. In ordered to understand how temperature influences amyloid fibril nucleation at the molecular level, we considered abovementioned three temperatures for the study. The radial distribution function, g(r) of water was determined for 4DEP simulations at 300, 333 and 353 K temperatures suggest that the characteristic structure of liquid water is preserved even at higher temperature (Figure S2). At 300 K, the Cα atom RMSD did not undergo much change and gave rise to a single Gaussian distribution with peak at about 4.0 Å, while Rg of backbone gave rise to two distributions with peaks at 9.5 and 8.7 Å suggesting formation of two compact globular structures (Figure 3A). The compact structures were held by hydrophobic association contributed by hydrophobic side-chains from all four strands (Figure S3). The cos(θ) angle between the



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chains C1 and C2 and between C3 and C4 indicated anti-parallel orientation while no parallel or anti-parallel orientation was observed between chains C2 and C3 at 300 K suggesting that the two dimeric β-sheets were intact in the compact structure (Figure 4A, 4DEP 300 K). Network layout at 300 K showed one type of conformation, which was compact and globular in nature (Figure S3, Figure 4B). The simulation did not result in a single-layered tetrameric β-sheet. The contact map shows partial contact between chains C1 and C4 and full length contact between chains C1 and C2 and between chains C3 and C4 (Figure S4, 4DEP 300 K). Nematic order shows a value less than 0.5 for the majority of conformations suggesting an absence of β-sheet (Figure S5). A second simulation at 300 K with different initial velocities also resulted in a similar kind of structure but was less compact (Figure S6, 4DEP 300 K). On the other hand, at 333 K, the RMSD and Rg showed a sharp transition at 112 ns, giving rise to two Gaussian distributions of different populations with RMSD showing peaks at 2.5 and 12 Å and Rg showing peaks at 9.5 and 11 Å (Figure 3B). The distribution with higher RMSD and Rg has an oblong structure where one dimer is displaced with respect to the other and is referred as staggered structure (Figure 3B; top panel snapshot at 50 ns). The second population with lower Rg showed a well ordered single-layered tetrameric β-sheet, square in shape and partially twisted, similar in construct with that of the reported amyloid seed14 (top panel Figure 3B, third snapshot onwards). Network layout also showed two clusters with population percentages of 94% (Cluster 1) and 6% (Cluster 2), corresponding to a single-layered tetrameric β-sheet and a staggered structure respectively (Figure 4B, 4DEP 333 K). Within the first 50 ns of simulation a staggered structure was formed which persisted till 111 ns, following which there was a sharp transition, accompanied by a 180◦ rotation of the C3-C4 dimer, with respect to the other dimer C1-C2. This is evident from the change in cos(θ) value from 1 to -1 at


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111 ns involving the β-strands C2 and C3 at 333 K, resulting in an anti-parallel orientation (Figure 4A, 4DEP 333 K). The β-strands C2 and C3 subsequently developed six to seven mainchain inter-strand hydrogen bonds which formed almost in a cooperative manner resulting into a single-layered tetrameric β-sheet, C1C2C3C4 , wherein the middle two β-strands C2 and C3 were anti-parallel and the outer two β-strands (C1-C2 and C3-C4) were parallel. This is in agreement with the observed contacts (involving C1-C2, C2-C3 and C3-C4) in the Cα contact map (Figure S4, 4DEP 333 K). The nematic order showed a value greater than 0.5 for the majority of conformations suggesting a well formed β-sheet structure (Figure S5, 4DEP 333 K). The singlelayered tetrameric β-sheet thus formed was stabilized by two salt-bridges, six hydrophobic side chain interactions (three in each face), four aromatic interactions (two in each face) and fourteen inter-strand hydrogen bonds (Figure 4C and Figure S7). The structure, once formed, was stable for the rest of the simulation time (2 µs). The simulation was performed thrice with different initial velocities and each time the resulting structure was a single-layered tetrameric β-sheet of the type C1C2C3C4, although the time of transition varies in these simulations. The second simulation of 4DEP 333 K showed a transition at about 50 ns (Figure S6). At 353 K, the RMSD showed three sharp transitions at 480, 770, 1500 ns (Figure 3C). The distribution of RMSD showed three peaks while backbone Rg showed two peaks implying that two of the three structures are of similar size. Network layout showed three distinct clusters identified as 1, 2 and 3 (Figure 4B, 4DEP 353 K) with 40%, 36% and 24% of populations, respectively. The clusters 1 and 2 showed up as ordered single-layered tetrameric β-sheet having all parallel (Cluster 1, C2C1C4C3) and mixed parallel and anti-parallel β-strands (Cluster 2, C2C1C4C3) respectively, while the cluster 3 showed up as a staggered conformation (Figure 4B, 4

DEP 353 K). During the first 16 ns, a single-layered staggered parallel β-sheet formed and



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persisted till 470 ns, after which there was a sharp transition accompanied by 180◦ rotation of C3C4 dimer with respect to the C1-C2 dimer, to form a single-layered tetrameric β-sheet (C2C1C4C3) wherein C1-C4 were anti-parallel and C2-C1 and C4-C3 were parallel. This is in agreement with the observed contacts (involving C1-C2, C1-C4 and C3-C4) in the contact map (Figure S4, 4DEP 353 K). This is also evident from the change in cos(θ) value from 1 to -1 at about 480 ns between C1 and C4 forming anti-parallel orientation (Figure 4A, 4DEP 353 K). The β-sheet C2C1C4C3 persisted till 743 ns and then there was a second transition at 770 ns during which the hydrogen bonds between C1 and C4 broke apart and the C3-C4 dimer underwent another 180◦ rotation, eventually forming a single-layered parallel tetrameric β-sheet (C2C1C4C3). This was consistent with the observed change in the cos(θ) value from -1 to 1 at 770 ns between C1 and C4 β-strands. Further, a third transition took place at about 1500 ns during which C1 and C4 broke again and the C3-C4 dimer underwent a 180◦ rotation with respect to the C1-C2 dimer, giving rise to a C2C1C4C3 β-sheet as observed earlier which has partially protruding C3 β-strand (Figure 3C top panel, 1900 ns snapshot). This is also accompanied by a change in cos(θ) value from 1 to -1 at 1500 ns. The nematic order shows a value greater than 0.5 for a majority of the time except in the transition regions suggesting the formation of different β-sheet structures (Figure S5, 4DEP 353 K). The most populated β-sheet thus obtained at 353 K was stabilized by one salt-bridge, two aromatic interactions, two hydrophobic side chain interactions and twelve inter-strand backbone hydrogen bonds. Thus, there were more stabilizing interactions and one transition at 333 K as compared to 353 K, suggesting that the higher temperature hindered the stability by reducing the number of interactions and increasing the number of transitions (Figure 4C). The second 4DEP 353 K simulation showed one transition and several sharp fluctuations in


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RMSD and Rg values eventually leading to a single-layered all parallel β-sheet (C1C2C4C3) (Figure S6).

MD simulation starting from many different starting structures converged into a single layered tetrameric β-sheet In the previous section, we observed transformation of a double layered dimer (4DEP) into a highly stable single-layered tetrameric β-sheet at 333 K. In addressing whether this highly stable structure is independent of any other starting structures, we performed three more simulations with different initial structures. In this endeavor, we first attempted a simulation wherein the four strands were placed in parallel and separated by 15 Å (identified as 4SEPa, Figure 1 and Figure 5A) to minimize the inter-strand interactions in the starting structure. During this simulation, within the first 100 ns, the four strands approached each other and formed a compact globular structure. This is accompanied by a significant drop in the Rg value from 14 to 9 Å (Figure 5A). The network cluster analysis performed on 4SEPa simulation resulted in three distinct clusters (Figure 6A, 4SEPa). The structures observed during the initial 100 ns were globular in shape and constitute Cluster 3 of the network layout with 12% of the population. The hydrophobic sidechains of Ile and Leu residues of all the four strands form the core of the globular structure, while polar side-chains are exposed to the solvent water (Figure 6A, Figure S8A, 4SEPa). The structure is further stabilized by several randomly orientated hydrogen bonds, which are located at the periphery of the globular structure. At around 290 ns, three strands (C1, C4 and C3) come together joined by several hydrogen bonds, while strand C2 was held only by hydrophobic interactions. Such structures formed Cluster 2 of the network layout with 34% of the population (Figure 6A). The structure persisted till 400 ns and thereafter the individual chains started to



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rearrange themselves by forming main-chain inter-strand hydrogen bonds to form a parallel C2C4C1 β-sheet at about 465 ns. Often, side-chain to main-chain inter-strand hydrogen bond precedes and directs the formation of inter-strand main-chain to main-chain hydrogen bond. At about 575 ns, the strand C3 started forming hydrogen bonds with already formed C2C4C1 β-sheet and thereby forming a single-layered tetrameric β-sheet (C2C4C1C3). This is evident from the observed change in cos(θ) value to -1 at about 600 ns (Figure S8B, 4SEPa). The contact map showed Cα-Cα contacts between the chains C1-C3, C2-C4 and C1-C4 (Figure S4, 4SEPa). Formation of tetrameric β-sheet is also evident from the nematic order plot where an average value of 0.8 is obtained after 600 ns (Figure S5, 4SEPa). Intriguingly, the structure thus formed remained stable for the rest of the simulation time (till 1 µs) and was stabilized by twenty mainchain hydrogen bonds, three salt-bridges and several hydrophobic interactions (Figure 6B, 4

SEPa). The process of such aggregation seems to be driven by the increase in solvent entropy,

which is accompanied by a decrease in the number of solvent-peptide hydrogen bonds and corresponding increase in peptide-peptide hydrogen bonds freeing several water molecules to the bulk water (Figure S9, 4SEPa). A similar, mechanism was reported in the assembly of several amyloid forming peptides50-51. Thus, in spite of a large inter-strand separation (15 Å) in the starting structure, the simulation again resulted in a single-layered tetrameric β-sheet. In the second run of 4SEPa simulation, a similar single-layered tetrameric β-sheet was formed (C3C1C2C4) but without going through an intermediate state (Figure S6). Another variant of a double-layered dimer structure wherein each strand was anti-parallel with respect to the adjacent ones (Figure 1, 4DEA), was subjected to MD simulation for 2 µs (Figure 5B). Network layout showed five distinct clusters. Within the first 55 ns of the simulation the four strands came close to each other and were held by several hydrogen bonds


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and hydrophobic interactions (Figure 6A, Cluster 5,4DEA). Subsequently, they formed a compact globular structure similar that obtained in. 4SEPa simulation where the inner core was formed by hydrophobic residues, mostly Leu, Ile and Tyr belonging to the four chains. These structures formed Cluster 2 with 21% of the population in the network layout. The individual strands associate with each other reducing the peptide conformational entropy and simultaneously increase the solvent entropy by freeing the hydrogen bonded water molecules to the bulk water. This is consistent with the observation of gradual decrease in peptide-solvent hydrogen bonds and corresponding increase in peptide-peptide hydrogen bonds (Figure S9, 4

DEA). At about 127.6 ns, the chains C1, C2 and C4 started to align with one another resulting in

C1C2C4 β-sheet. The orientation of the β-strands, parallel or anti-parallel, could be readily correlated with the cos(θ) versus simulation time plot (Figure S8B, 4DEA). Thereafter, at about 254 ns, the lone chain C3 started aligning with the already formed C1C2C4 β-sheet with an antiparallel orientation resulting in a single layered tetrameric β-sheet (C1C2C4C3) with protruding C3 strand at about 663 ns (Cluster 3, Figure 6A, 4DEA). This structure persisted till 940 ns, for a duration of about 230 ns. Thereafter, the strand C3 took a sharp 180◦ rotation, as is evident from the change in cos(θ) value of C4 and C3 from -1 to 1, at about 960 ns (Figure S8, 4DEA). Thus, all the four strands end up in a twisted square like single-layered tetrameric β-sheet of the type C1C2C4C3. This structure turned out to be the most populated (53%) cluster (Cluster 1) and possessed ~20 inter-strand main chain hydrogen bonds, one salt-bridge and several hydrophobic interactions (Figure 6B, 4DEA). The contact map showed Cα contacts between the chains C1-C2, C2-C4 and C3-C4 (Figure S4, 4DEA). Formation of a tetrameric β-sheet is also seen from the nematic order plot after 800 ns (Figure S5, 4DEA). The Cluster 1 of 4DEA simulation, which is a single-layered tetrameric β-sheet, looks similar to the Cluster 1 of 4SEPa simulation (Figure 6A).



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In the second run of 4DEA simulation, we observed formation of a non-planar anti-parallel single-layered tetrameric β-sheet (C2C1C3C4). It formed without going through an intermediate state as shown in the Figure S6. In yet another variant of the starting structure, the double-layered dimer (4DHP), in which each monomeric unit was chosen to be in a β-hairpin conformation (Figure 1). In this simulation, each of the β-hairpins in the double layered dimer started to unfold one after another in a sequential manner as can be seen from the DSSP plot (Figure S10). The first one (C1) unfolded at about 22 ns into an extended structure and served as a template to which the second unfolding hairpin (C2) paired by hydrogen bonding in an anti-parallel orientation (i.e. C1C2). The unfolding of β-hairpins, C2 and C3, took place almost simultaneously at about 47.4 and 47.6 ns, respectively, while C4 β-hairpin unfolded at about 61 ns (Figure 5C, snapshot at 47.6 ns). After the unfolding of the hairpins, chains C3 and C4 sample partially bent structures till 209.5 ns, as is evident from the DSSP plot (Figure S10). Thereafter, the bent structures of C3 and C4 transformed into β-strands and then the β-strand C4 started forming hydrogen bonds with the βstrand C2, giving rise to a trimeric β-sheet (C1C2C4). During this time the β-strand C3 started to associate with C4 and this structure can be seen in Cluster 2 (Figure 6A, 4DHP). Finally, at about 420 ns, it resulted in the formation of a single-layered tetrameric β-sheet of the type C1C2C4C3 which has partially protruding C1 strand (Cluster 1 of 4DHP, Figure 6A). The cos(θ) plots showed that C1-C2 and C3-C4 are anti-parallel and C2-C4 are parallel with respect to each another (Figure S8, 4DHP). The Cα contact map showed contacts between the chains C1-C2, C2C4 and C3-C4 (Figure S4, 4DHP). Formation of tetrameric β-sheet was seen from the nematic order plot after 400 ns (Figure S5, 4DHP). Although, the β-sheet was observed for the 4DHP simulation, it was not well ordered as can be seen by the observed fluctuations in the P2 values.


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The single-layered tetrameric β-sheet thus formed was stabilized by ~14 inter-strand hydrogen bonds, one salt-bridge, one hydrophobic interaction and two aromatic interactions. Intriguingly, this structure (Cluster1 of 4DHP) showed resemblance to the centroid structure of Cluster 3 of 4

DEA simulation in its β-strand orientation (Figure 6A). The second run of 4DHP simulation did

not form a single layered tetrameric β-sheet during the 1 µs of simulation time. It showed unfolding of a β-hairpin during the first 200 ns and formation of anti-parallel dimer β-sheet C3C4 at about 700 ns during which chain C2 was extended while chain C1 was still sampling a βhairpin conformation (Figure S6). This simulation is not converged yet and might take more time to form a tetrameric β-sheet. Thus, four different starting structures resulted in a similar singlelayered tetrameric β-sheet having mixed parallel and anti-parallel β-strands at 333 K, asserting the unique propensity of the sequence to form a highly ordered β-sheet structure.

β-Sheet with mixed parallel and anti-parallel β-strands, forms stable higher order multilayered oligomers Can the stable single-layered tetrameric β-sheet structure serve as the basic nucleation unit for the amyloid fibril formation? In addressing, whether such a unit can lead to higher order fibrilar oligomer, we further carried out MD simulations on multi-layered structures having 6, 8, 16 monomeric units (6DEP, 8DEP, 8DEAm, 16FEP and 16FEAm) (Figure 1). We presumed that the double-layered structures may form a single-layered β-sheet as observed in case of 4DEP simulation at 333 K and therefore we performed MD simulations with 6

DEP and 8DEP as starting structures. During the simulation, the Rg and RMSD parameters did

not change much and the double-layered structure remained intact for 6DEP and 8DEP simulations (Figure 7A, B). We observed several contacts between the chains from opposite



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layers (Figure S4). P2 of 6DEP was well below 0.5 suggesting a relatively disordered doublelayered β-sheet while for 8DEP simulation it was fluctuating around 0.5 suggesting a partially formed doubled-layered β-sheet. Network layout generates single cluster for each of the simulations (Figure S11, 6DEP and 8DEP). The centroid structures of 6DEP and 8DEP simulations showed two to three saltbridges between Glu and Lys side chains from opposite βsheets which were often bridged by Tyr side-chain by hydrogen bonding (Figure S12). In both 6

DEP and 8DEP simulations, we observed an angular orientation between the opposite β-sheets

due to longer interacting side chains (Glu and Lys) at one side and compaction at the other side due to hydrophobic association involving Ile, Leu and Phe. We observed an average angle of 70.8±8.0˚ between the β-sheets in 6DEP simulation and an angle of 44.5±12.0˚ in 8DEP simulation (Table S2). The average inter β-sheet distances were 18.0±0.8 Å (6DEP simulation) and 16.1±0.6 Å (8DEP simulation), in spite of the starting structures being parallel and having crystallographic distance of 10 Å (Figure S13). The second simulations of 6DEP and 8DEP result in similar conformational types (Figure S6). Non-observation of single-layered hexameric and octameric β-sheets in 6DEP and 8DEP simulations could be due to high entropic cost to align all the six and eight strands in one plane. Such structures having large angular orientation between the β-sheets and large inter β-sheet distance may not lead to an ordered β-sheet structure required for propagation of amyloid fibril. In the above two simulations, the starting structures were built using only parallel strands. Following this, we chose the stable single-layered tetrameric β-sheet (C1C2C3C4) that resulted from the 4DEP 333 K simulation as a template to construct a two-layered and a four-layered tetrameric β-sheet structures. (Figure 1, 8DEAm and

16

FEAm). Intriguingly the double-layered

tetrameric β-sheet structure in 8DEAm simulation was found to be highly stable during the entire


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period of simulation resulting in unimodal distributions of RMSD and Rg (Figure 7C). Several contacts were observed between the chains of opposite layers (Figure S4) and P2 is well above 0.5 suggesting ordered β-sheet structure (Figure S5). The network layout resulted into a single cluster (Figure S11) which has 2 to 3 inter-β-sheet saltbridges as observed in 6DEP and 8DEP simulations (Figure S11, Figure S12 8DEAm). We observed only a slight angular orientation of about ~145.7±5.8◦ between the opposite β-sheets and an inter β-sheet distance of 13.9±0.5 Å which was found to be much smaller than the previous two cases (in 6DEP and 8DEP simulations) where the distances were 18.0±0.8 and 16.1±0.6 Å respectively (Figure S13). Hence, there is a greater possibility for such a structure to further grow into amyloid fibril due to shorter inter-β-sheet distances. Further, to assess the feasibility of higher order oligomers and to study their stability and dynamics, we performed two more simulations (16FEP and

16

FEAm) on four layered structures

with four strands in each layer. In the former case (16FEP) all the strands were parallel to each other, while in the latter case the four layers were built from a β-sheet (C1C2C3C4) having mixed parallel and anti-parallel β-strands as mentioned earlier (Figure 1). In

16

FEP simulation, the

structure started to break down immediately after the start of simulation suggesting instability of higher ordered structure when built from all parallel β-strands which could be due to the onset of angular orientation between sheets as the simulation begins. Both RMSD and Rg were changing as a function of time with Rg showing a rising trend indicating larger sizes of the resultant structures (Figure 7D). There were several missing Cα contacts and P2 was almost zero after 450 ns (Figures S4 and S5). The network layout showed three clusters and their centroids showed irregular β-sheet structures which were more like an amorphous aggregate (Figure S11). Such structures would be unable to propagate into ordered fibrilar structures. On the other hand, the



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simulation with

16

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FEAm starting structure, resulted in a highly stable ordered four-layered β-

sheet structure having unimodal Rg and RMSD distributions (Figure 7E). There were several Cα contacts between the adjacent layers (Figure S4) and P2 was well above 0.5 (Figure S5) for the entire duration of the simulation. The network layout showed a single cluster (Figure 11). The structural stability of the multi-layered β-sheet structure in

16

FEAm was evident from the three

inter β-sheet distances, which are 13.5±0.5, 12.1±0.4 and 13.4±0.4 Å for 1st-2nd, 2nd-3rd and 3rd4th β-sheets respectively (Figure S13). The central innermost inter β-sheet distance (12.1±0.4 Å) between 2nd and 3rd β-sheets was smaller than the outer two (13.5±0.5 and 13.4±0.4 Å) suggesting desolvation of the innermost β-sheets which strengthens their interactions.

The

desolvation of outer two β-sheets was less as one of the interacting sides is the inner β-sheet while the other side is water. This is confirmed by observation of less number of peptide-solvent hydrogen bonds for the innermost β-sheets (37±3.1 and 38±2.9 for β-sheets 2 and 3) as compared to the outer ones (42±3.2 and 41± 3.1 for β-sheets 1 and 4) (Figure S14). Intriguingly, the inter βsheet distance (between 2nd and 3rd β-sheets) is close to the crystallographic distance of 10 reported for amyloid fibrils16,

35

. Similar interactions as observed in 8DEAm were found to

stabilize the oligomeric structure observed in 16FEAm simulation (Figure S15). Therefore, these simulations suggest that a stack made up of mixed parallel and anti-parallel β-sheets provide sufficient attachment sites for inter β-sheet interactions to further propagate into a fibrilar structure.

Conformational potential energies correlate well with the number of hydrogen bonds The average potential energies were calculated for the predominant conformers of the selected simulations (Table 1). Averaging was performed by considering the potential energy of a


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continuous stretch, which is sampling a specific type of conformation. Considering the fact that the simulations described above differ only in their starting structure and in their oligomeric state, the average potential energies were estimated and compared between the conformers having equal number of amino acid residues. If we consider a given type of conformation resulting from similar number of residues for example, the single-layer tetrameric β-sheets are expected to have same entropic contribution, the conformation having the lowest potential energy will have lowest free energy and therefore higher stability. Based on this criterion, conformations indicated in bold in Table 1 are defined as stable structures. From this table, we could infer that there is a strong correlation between the average potential energy and the average number of main-chain hydrogen bonds with a correlation coefficient of -0.98 (Table 1). This is expected as the hydrogen bonds are the major contributing factors in the amyloid fibril formation. Figure S16 shows that both the hydrogen bonds and hydrophobic contacts are optimumal in 4DEP 333 K simulation. The average potential energies of all the tetrameric forms could be categorized under two potential energy ranges such as -2530 to -2607 kJ/mol for squarish single-layered tetrameric βsheet (Table 1, in bold) and -2325 to -2431 kJ/mol for staggered conformations observed in 30100 ns stretch at 333 K. (Table 1). Staggered conformations have less number of interactions and therefore possess higher potential energy. The potential energies of the conformations observed in 8DEP and 8DEAm simulations are comparable in spite of structural differences suggesting similar number and types of interactions. On the other hand, the potential energy difference between the multi-layered structures

16

FEP and

16

FEAm was ~559 kJ/mol, suggesting a

significant difference (energy equivalent of ~28 hydrogen bonds) arises due to greater stability of the conformation in the case of 16FEAm as compared to 16FEP.



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DISCUSSION Optimum temperature for the formation of a single layered tetrameric β-sheet is 333 K Nature has adopted certain mechanisms such as cooperative folding, folding in the presence of chaperone, and under physiological pH and temperature to correctly fold the polypeptide chain into its native state. However, disruptions in one or more of these factors can lead the polypeptide to misfold or adopt an alternative conformation such as amyloid fibril8. The experimental studies suggest that the amyloid fibrils formed from apo-myoglobin or from the Ghelix peptide are fundamentally similar to the diseased state of amyloid fibrils and occur at a higher temperature35. In the present study, we monitored the effect of temperature on the formation and stability of highly-ordered single-layered tetrameric β-sheet. At 333 K temperature starting from 4DEP, we observed 180◦ flipping of the C3-C4 dimer with respect to the C1-C2 dimer, which gives rise to a well ordered single-layered tetrameric β-sheet (C1C2C3C4). It was stable for 1.9 µs in a 2 µs simulation run. Such strand flipping was observed only at higher temperatures (333 and 353 K). At 353 K, we observed faster strand flipping and therefore many transitions during the course of simulation leading to less stable tetrameric β-sheet structure. Non-observance of the strand flipping at lower temperature (300 K) could be attributed to the structure being trapped in a local free-energy minimum which would require an additional free energy to overcome the barrier to form a single-layered tetrameric β-sheet. Higher temperatures lower the free energy barrier and therefore enhances the probability of sampling high-energy conformations. Therefore, the initial step of nucleation which is a rate limiting step is facilitated at higher temperatures. The study thus reveals that optimum temperature required for the formation of a stable single-layered tetrameric β-sheet is 333 K.


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Single-layered tetrameric β-sheet is the basic nucleation unit formed through multiple pathways The nucleation process, in the amyloid fibril formation has been shown to be thermodynamically unfavorable14, 52. However, once the nucleus is formed further propagation into amyloid fibril is thermodynamically favorable. Therefore, the critical step in the amyloidogenesis is the nucleation. While performing MD simulations on a variety of initial configurations (4DEP, 4

SEPa, 4DEA and 4DHP), we observed formation of similar single-layered tetrameric β-sheets.

The Cα-root-mean-square fluctuation showed flexibility near the N- and C- termini as compared to the interior residues, a characteristic pattern seen in peptides forming amyloid fibril14 (Figure S18). Such structures are known to be potential candidates for amyloid fibril formation due to their high structural stability and exposed hydrophobic, polar and charged side-chains on both faces of the β-sheet to further interact with other such β-sheet. Therefore, such structural units can serve as nucleation unit or seed in amyloid fibril propagation. Formation of amyloid seed consisting of three to four β-strands is also reported in other studies14,

28

. For example, MD

simulations performed on human calcitonin-derived peptide DFNKF oligomer, where parallel dimer, trimer and tetramer strands were simulated for 10 ns, using CHARMM-22 at 350 K, revealed that the structural stability increases in going from dimeric to tetrameric state14. Another MD simulation performed on a seven residues long yeast prion sup-35 fragment (GNNQQNY), which was also performed only for 10 ns, concluded that the minimal requirement for the fibrilar seed formation could be three or four peptides28. In yet another simulation study on KFFE peptide, which is prone to amyloid fibril formation, showed formation of dimers, trimers and tetramers with different chain orientations and they are in rapid equilibrium with each other27.



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All these studies revealed a smaller size of the seed for amyloid fibril formation as is the case with our simulations too. The β-sheet percentage was estimated for each of the simulations (Figure S19). For many simulations the value is constant while for others, it stabilizes after initial few hundreds of ns in 4SEPa, 4DEA, 4DHP simulations. The Cα RMSD also follows a similar trend (Figures 3, 5, 7, S6). Steady values of both these parameters indicate convergence of the simulations. Proteins or peptides, which are not amyloidogenic can also form amyloid fibrils under certain conditions, which implies that there are certain common pathways that operate irrespective of whether a peptide or protein is amyloidogenic or non-amyloidogenic. In our simulations we observed three distinct pathways for the formation of a single-layered tetrameric β-sheet structure. According to the first pathway, nucleation takes place by a sharp transition without formation of any intermediate, as in the case of 4DEP 333 K simulation. The sharp transition is brought about by cooperative formation of hydrogen bonds53. In the second pathway, nucleation takes place through an intermediate state. In 4DEA and 4SEPa simulations, the formation of the single-layered tetrameric β-sheet preceded by the formation of a compact globule like intermediate state, which persisted for a duration of 427 and 463 ns, respectively. The formation of a globular structure or coalescence of hydrophobic residues was a fast process while the re-arrangement within the globule turned out to be a relatively slower process. In the third pathway, the single-layered tetrameric β-sheet structure has formed by dock and lock mechanism14,

54

. For example, in 4DHP simulation, loosely held β-hairpins unfolded into an

extended conformation one after the other and formed a tetrameric β-sheet, which has the potential to serve as an amyloid nucleus. A similar mechanism was reported for amyloid β (2535) peptide where β-hairpin undergoes a transition to extended β-strand and in the final fibril


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structure, β-hairpin was absent55. Recently, it was reported that the β-hairpin conformation in the amyloid β peptide slows down the aggregation process56-57. In our study, non-observation of βhairpin in the aggregated structure could be due to the intramolecular interactions of the backbone atoms which are not free to interact with other monomeric peptides. On other hand more sites are available for intermolecular interactions in extended conformation. Taken together, we believe that the initial phase of nucleation follows multiple pathways to reach a similar stable state. Although, the overall mechanism of fibril nucleation seems to be similar with the reported studies3, 51, 58-59, the subtle differences observed during the process are distinct and unique to the current sequence. Whether, a peptide follows one or the other pathways depends upon several factors such as concentration, temperature and hydrophobic exposure58,

60

.

Hydrophobic exposure depends upon the conformational form of the peptides in the oligomer. For example, in 4DEP simulation at 300 K, the hydrophobic exposure is less due to burial of the hydrophobic side-chains in the core of the compact globule which led to an amorphous like aggregate (Figure 3A). On the other hand, the hydrophobic side-chains which were more exposed in 4DEP simulation at 333 K due to temperature induced dynamics of the peptides, led in the direct formation of ordered structures without going through any intermediate state. This is in line with the studies of Cheon et al. 58, which revealed that exposure of hydrophobic residues promotes fibrilar structure while their burial favors an amorphous aggregate. In two of our simulations (4SEPa and 4DEA), formation of intermediates was observed even at 333 K, primarily because of the least exposure of hydrophobic side-chains in the initial state of collapse. Our study thus provides mechanistic details of the amyloid fibril nucleation. Some of the mechanisms of fibril nucleation observed in our study overlap with the prior studies27, 58, 61. For example, the Aβ25-35 (GSNKGAIIGLM) and Aβ16-22 (KLVFFAE) peptides follow the first



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(without intermediate) and the second (through an intermediate) pathways, respectively, prior to the amyloid fibril formation as one peptide is polar and the other is highly hydrophobic58. The second pathway which involved in the formation of an intermediate is observed in KTVIIE61, KFFE27 and Aβ(16-22) 62 peptide fragments. The third pathway where each individual β-strand acts as a template is in line with the MD simulation carried out earlier on human calcitonin-derived peptide DFNKF, which resulted in a parallel oligomer14. A mechanism similar to this was observed for the insulin peptide hormone, LVEALYL where fibril formation takes place through dock and lock mechanism54.

The higher order assembly mechanism in amyloidogenesis This study provides a framework for the formation of higher order oligomers. We observed polymorphic behavior of the Ac-IKYLEFIS-NMe peptide in its multimeric state. The adopted conformations showed variety of tetrameric conformations where the β-strands orientations are different, resulting in different types of stable tetrameric β-sheet structures, with mixed parallel and anti-parallel β-strands. Such polymorphism and high degree of plasticity is reported in the case of GNNQQNY peptide from yeast prion protein Sup3563. X-ray diffraction and solid-state NMR experiments also reveal occurrence of such polymorphic fibrils64. All of these β-sheets have the potential to aggregate into amyloid fibril as they possess several interacting sites. The simulation of 16-mers (16FEAm) gives rise to a highly stable multi-layered structure which could further aggregate into amyloid-like fibril. In one case we could successfully model the formation of a multi-layered fibril-like structure.


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Driving forces for amyloid fibril formation and stability The early stage of amyloid fibril formation involves nucleation as the initial event, which in our case commences with the formation of a single-layered tetrameric β-sheet. These β-sheets have the tendency to stack upon each other and form a multi-layered oligomeric structure, which eventually gives rise to amyloid fibril. The single-layered tetrameric β-sheet obtained at 333 K from various simulations always possessed mixed parallel and anti-parallel β-strands but never had all parallel strands, except at a very high temperature (353 K). The anti-parallel stands are primarily linked by hydrogen bonding, hydrophobic interactions and saltbridges, while parallel strands are mostly held by hydrogen bonding and hydrophobic interactions. Rarely, saltbridges were seen to stabilize the parallel β-strands in a β-sheet. Hydrogen bonds are the predominant interactions in stabilizing the amyloid fibril. In parallel β-strands hydrogen bonds were expected to be weaker than the anti-parallel β-strands because of their angular orientation. Therefore, βsheet with mixed parallel and anti-parallel β-strands turned out to be more stable structure and is observed frequently in our simulations. Further, in the mixed parallel and anti-parallel arrangement, the charged residues Arg and Glu are oppositely orientated and favor inter-β-sheet saltbridge interactions as observed in case of 8DEAm and

16

FEAm simulations. The hydrogen

bonding, hydrophobic and aromatic interactions are optimal in that arrangement. The protruding sticky sites such as charged and hydrophobic side chains above and below the single layered tetrameric β-sheet provide sites for stacking interactions with other such β-sheets and thus facilitate propagation into amyloid fibril. Similar types of interactions were also reported for the formation and stability of amyloid fibril formed by the peptides differing in sequence composition and length14, 21, 28, 30, 50, 65.



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A short sequence with amyloid propensity can nucleate fibril formation as a part of fulllength protein Several neurodegenerative diseases are linked to aggregation of amyloid fibril. In a protein not all parts are important for initiating protein folding. Similarly, not all parts are important for initiating protein aggregation. There are certain preferred sequences (one or more) normally present in amyloid fibril forming proteins and they drive the fibril formation of the whole protein 1, 64

. Recently, many proteins have been identified to have 4-12 residues segments which

independently form amyloid fibrils 1, 64. In an α/β protein acylphosphatase, there are two distinct regions, which are rich in hydrophobic residues. They have β-sheet propensity and are responsible for aggregation, while other sequence regions are important for folding1. In a denatured state the hydrophobic residues are solvent exposed and can readily form intermolecular interactions and can thus lead to aggregation. On the other hand, the myoglobin protein is not amyloidogenic under physiological conditions. However, when incubated at 60 ◦C the full-length protein and the G-helix alone formed amyloid fibrils 35. This could be because of preferred amyloid fibril forming sequence IKYLEFIS. Such a sequence eventually can drive the aggregation of whole protein where the studied region might stack upon one another leaving the folded part dangling out of the fibrilar aggregate.

Conclusions Overall, the current study on IKYLEFIS peptide from the G-helix of myoglobin provides mechanistic details of amyloid fibril formation which can be extrapolated to understand fibril formation in other peptides and proteins. A higher temperature of 333 K turned out to be crucial for amyloid fibril nucleation. The single-layer tetrameric β-sheet serves as a nucleating unit to


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propagate aggregation. It formed through three distinctive pathways, with or without an intermediate or by dock and lock mechanism. The single-layer tetrameric β-sheets showed a polymorphic behavior. It provides sites for stacking interactions. The stability of a multi-layered β-sheet destined to form amyloid fibril was governed by an interplay of both intra- and inter-βsheet interactions where hydrogen bonding, saltbridges, hydrophobic, and aromatic interactions are the major contributors.

Acknowledgment We thank TCIS, Hyderabad Supercomputing Facility. KVRC thanks Department of Science and Technology, Government of India, New Delhi, for his JC Bose National Fellowship (DST, GoI).

Supporting Information The supporting information contains part of Methods, a short summary of the Results and additional figures to substantiate the content.

References (1) Chiti, F.; Taddei, N.; Baroni, F.; Capanni, C.; Stefani, M.; Ramponi, G.; Dobson, C. M., Kinetic partitioning of protein folding and aggregation. Nat. Struct. Mol. Biol. 2002, 9, 137-143. (2) Dobson, C. M., Principles of protein folding, misfolding and aggregation. Sem. Cell Develop. Biol. 2004, 15, 3-16. (3) Murphy, R. M., Peptide aggregation in neurodegenerative disease. Annu. Rev. Biomed. Eng. 2002, 4, 155-174. (4) Chiti, F.; Dobson, C. M., Amyloid formation by globular proteins under native conditions. Nat. Chem. Biol. 2009, 5, 15-22.



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(5) Eisenberg, D.; Jucker, M., The amyloid state of proteins in human diseases. Cell 2012, 148, 1188-1203. (6) Knowles, T. P. J.; Vendruscolo, M.; Dobson, C. M., The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell. Biol. 2014, 15, 496-496. (7) McParland, V. J.; Kad, N. M.; Kalverda, A. P.; Brown, A.; Kirwin-Jones, P.; Hunter, M. G.; Sunde, M.; Radford, S. E., Partially unfolded states of beta2-microglobulin and amyloid formation in vitro. Biochemistry 2000, 39, 8735-8746. (8) Fandrich, M.; Fletcher, M. A.; Dobson, C. M., Amyloid fibrils from muscle myoglobin. Nature 2001, 410, 165-166. (9) De Felice, F. G.; Vieira, M. N. N.; Meirelles, M. N. L.; Morozova-Roche, L. A.; Dobson, C. M.; Ferreira, S. T., Formation of amyloid aggregates from human lysozyme and its diseaseassociated variants using hydrostatic pressure. The FASEB J. 2004, 18, 1099-1101. (10) Schmittschmitt, J. P.; Scholtz, J. M., The role of protein stability, solubility, and net charge in amyloid fibril formation. Protein Sci. 2003, 12, 2374-2378. (11) Pepys, M. B.; Hawkins, P. N.; Booth, D. R.; Vigushin, D. M.; Tennent, G. A.; Soutar, A. K.; Totty, N.; Nguyen, O.; Blake, C. C. F.; Terry, C. J., et al., Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 1993, 362, 553-557. (12) Carballo-Pacheco, M.; Strodel, B., Advances in the simulation of protein aggregation at the atomistic scale. J. Phys. Chem. B 2016, 120, 2991-2999. (13) Iannuzzi, C.; Maritato, R.; Irace, G.; Sirangelo, I., Misfolding and amyloid aggregation of apomyoglobin. Int. J. Mol. Sci. 2013, 14, 14287-14300.


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Table 1 Calculated average potential energies of a continuous stretch sampling specific type of conformation in distinct simulations. The potential energies of 4-mers, 8-mers and 16-mers are shown in white, light gray and dark gray shades. The conformations of a given time stretch can be seen in Figure 3, 5 and 7. The conformers having lower potential energy are shown in bold.

Simulation types

Temperature (K) 300 333

4

DEP 353

4

SEPa DEA 4 DHP 8 DEP 8 DEAm 16 FEP 16 FEAm 4

333 333 333 333 333 333 333

Time segment (ns) 700-1000 30-100 200-1000 50-400 500-730 800-1000 600-1000 1200-2000 800-1000 500-700 300-700 500-1000 500-1000

Potential energy (kJ/mol) -2425.5±82.6 -2431.1±98.3 -2601.8±98.0 -2325.3±96.0 -2541.6±98.5 -2543.8±112.7 -2607.7±101.4 -2528.1±86.3 -2529.9±94.7 -5704.4±193.2 -5807.7±151.1 -11625.1±237.3 -12184.2±216.8

No. of backbone H-bonds 3-9 5-11 6-12 4-10 5-11 4-12 6-12 5-11 5-11 14-26 13-23 28-38 30-44


No. of total Hbonds 5-11 5-13 6-14 4-10 5-13 5-13 6-14 6-14 6-12 17-29 17-27 31-47 38-54


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Figure 1. An overview of the starting structures used in various MD simulations. Abbreviations, 1

Hx: Monomer in Helix, 4DEP: 4 monomers in Double-layers Extended Parallel, 4SEPa: 4

monomers in Square Extended Parallel Apart, 4DEA: 4 monomers in Double-layers Extended Anti-parallel, 4DHP: 4 monomers in Double-layers Hairpin Parallel, 6DEP: 6 monomers in Double-layers Extended Parallel, 8DEP: 8 monomers in Double-layers Extended Parallel, 8

DEAm: 8 monomers in Double-layers Extended Anti-parallel in Middle two strands, 16FEP: 16

monomers in Four-layers Extended Parallel and 16FEAm: 16 monomers in Four-layers Extended Anti-parallel in Middle two-strands. The direction of the β-strands in oligomer are indicated by N for N-terminal end. For multiple β-strands pointing to the same direction is indicated by one N. The stable structure obtained from 4DEP 333 K simulation was used to construct 8DEAm and 16

FEAm starting structures and are indicated in dotted arrow. 38 ACS Paragon Plus Environment

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Figure 2. Network cluster layouts of the trajectories on the monomeric peptide starting from native α-helical conformation at 300 and 333 K. A pairwise RMSD cutoff of 3.5 Å was used to build both the network layouts.



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Figure 3. Variation in Cα RMSD and Rg of backbone as a function of simulation time for the 4

DEP simulations performed at 300 K (A), 333 K (B) and 353 K (C) temperatures. The Cα

RMSD was calculated with respect to end structure of the trajectory. Running averages over 100 data points were taken for clarity on the RMSD and Rg plots (black). The normalized probability distributions of RMSD and Rg are shown in panels right-hand-side of the RMSD and Rg plots. Selected conformational states in cartoon representation and side-chains in lines at specific simulation times (ns) are shown on top of the RMSD panel and are indicated with dotted arrows. The peptide chains are indicated as C1 (red), C2 (green), C3 (blue) and C4 (pink).



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Figure 4. (A) Cos(θ) angle between two adjacent strands as a function of simulation time. (B) Network cluster layouts of 4DEP simulations at three different temperatures (300, 333 and 353


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K). (C) Various interactions stabilizing the single-layered tetrameric β-sheet conformation seen during 4DEP simulation at 333 K (centroid 1). All side chains are shown in stick representation, the salt-bridges are in red rectangle, hydrophobic interactions in grey rectangle, and the aromatic interactions in ellipse. For network cluster layouts, a 7.5 Å pairwise RMSD cutoff was used. The cluster percentages are shown along the side and the centroid structure of the cluster is indicated by an arrow. The peptide chains are indicated as C1 (red), C2 (green), C3 (blue) and C4 (pink).



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Figure 5. Variation in Cα RMSD and Rg of backbone as a function of simulation time in 4SEPa (A), 4DEA (B), 4DHP (C) at 333 K. The Cα RMSD was calculated with respect to end structure of the trajectory. Running averages over 100 data points were taken for clarity on the RMSD and Rg plots (black). The normalized probability distributions of RMSD and Rg are shown in panels right hand side of the RMSD and Rg plots. Selected conformational states at specific simulation times (ns) shown with dotted arrows are shown on top of the RMSD panel. Structures are shown in cartoon representation while side-chains are shown in lines. The peptide chains for the first and last structures in the top panel are indicated as C1 (red), C2 (green), C3 (blue) and C4 (pink).



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Figure 6. (A) Network cluster layouts of 4SEPa, 4DEA and 4DHP simulations. (B) Various interactions stabilizing the single-layered tetrameric β-sheet conformation seen during 4SEPa (centroid 1) and 4DEA (centroid 1) simulations. All side chains are shown in stick representation, the salt-bridges are in red rectangle, hydrophobic interactions in black rectangles. For network cluster layouts, a 7.5, 5 and 5 Å pairwise RMSD cutoffs were used for 4SEPa, 4DEA and 4DHP, respectively. The cluster percentages are shown along the side and the centroid structure of the cluster is indicated by an arrow.


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Figure 7. Variation in Cα RMSD and Rg of backbone as a function of simulation time during 6

DEP (A), 8DEP (B), 8DEAm (C),

16

FEP (D) and

16

FEAm (E) simulations at 333 K. The Cα



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RMSD was calculated with respect to end structure of the trajectory. Running averages over 100 data points were taken for clarity on the RMSD and Rg plots (black). The normalized probability distributions of RMSD and Rg are shown in panels right-hand side of the RMSD and Rg plots. Selected conformational states at specific simulation times (ns) shown with dotted arrows are shown on top of the RMSD panel. Structures are shown in cartoon representation while sidechains are shown in lines. The layers are indicated in red (layer 1), green (layer 2), blue (layer 3) and pink (layer 4).


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Table of Contents Image


1 Mechanism of Initiation, Association and Formation of Amyloid Fibrils

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