Assemblies of amyloid-β30–36 hexamer and its G33V/L34T mutants by replica-exchange molecular dynamics simulation
Assemblies of amyloid-β30±36 hexamer and its G33V/L34T mutants by replica-exchange molecular dynamics simulation
Zhenyu Qian 0 1
Qingwen Zhang 1
Yu Liu 0 1
Peijie Chen 0 1
0 Key Laboratory of Exercise and Health Sciences (Ministry of Education) and School of Kinesiology, Shanghai University of Sport , Shanghai , China , 2 College of Physical Education and Training, Shanghai University of Sport , Shanghai , China
1 Editor: Human Rezaei, INRA Centre de Jouy-en- Josas , FRANCE
The aggregation of amyloid-β peptides is associated with the pathogenesis of Alzheimer's disease, in which the 30±36 fragments play an important part as a fiber-forming hydrophobic region. The fibrillar structure of Aβ30±36 has been detected by means of X-ray diffraction, but its oligomeric structural determination, biophysical characterization, and pathological mechanism remain elusive. In this study, we have investigated the structures of Aβ30±36 hexamer as well as its G33V and L34T mutants in explicit water environment using replica-exchange molecular dynamics (REMD) simulations. Our results show that the wild-type (WT) Aβ30±36 hexamer has a preference to form β-barrel and bilayer β-sheet conformations, while the G33V or L34T mutation disrupts the β-barrel structures: the G33V mutant is homogenized to adopt β-sheet-rich bilayers, and the structures of L34T mutant on the contrary get more diverse. The hydrophobic interaction plays a critical role in the formation and stability of oligomeric assemblies among all the three systems. In addition, the substitution of G33 by V reduces the β-sheet content in the most populated conformations of Aβ30±36 oligomers through a steric effect. The L34T mutation disturbs the interpeptide hydrogen bonding network, and results in the increased coil content and morphological diversity. Our REMD runs provide structural details of WT and G33V/L34T mutant Aβ30±36 oligomers, and molecular insight into the aggregation mechanism, which will be helpful for designing novel inhibitors or amyloid-based materials.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by the China
Postdoctoral Science Foundation (grant no.:
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Alzheimer's disease (AD), characterized by cerebral extracellular amyloid plaques, is
agerelated and quite common among the senior population. Its pathogenesis is associated with
the accumulation of amyloid β-peptide (Aβ) and τ-protein [
], and evidences from genetics
and pathology support that the former trigger the pathogenesis process [
Aβmonomer is mainly disordered, while the major constituents of amyloid plaques display a cross-β
]. The mature amyloid fibril is believed to associate with neurologic degeneration
. However, converging studies suggest that small intermediate oligomers are the major
species responsible for neurotoxicity and dementia, which are formed in the early stage of
The Aβ peptide (40±42−amino acid) is the most abundant forms of Aβ in vivo, derived
from the amyloid precursor protein (APP) through proteolytic cleavage by β- and γ-secretase
]. In 2005, a β1-strand±turn±β2-strand motif was proposed to describe the Aβ1±42 fibrillar
structure using nuclear magnetic resonance (NMR), which contains two in-register β-sheets
that are formed by residues 18±26 and 31±42 [
]. Thereafter, another model for Aβ amyloid
with a similar motif was proposed, known as the Ma-Nussinov-Tycko model [
strandbend-strand structures were also resolved in Aβ1±40 fibrils with β-strand secondary structure
in residues 11±22 and 30±39, based on numerous constraints from solid-state NMR and
electron microscopy (EM) [
]. In 2013, a 3-fold structure of brain-derived Aβ1±40 fibrils was
detected by Tycko group through solid-state NMR [
]. The fibrils from two AD patients
display a distinct morphology. The determined molecular model includes a twist in residues 19±
23, a kink at G33, and a bend in glycine residues 37 and 38. Recently, a structure of an Aβ1±42
fibril composed of two twisted protofilaments was resolved by cryoelectron microscopy
]. The atomic model is comprised of β-strands in residues 1±9, 11±20, and 27±33, with
a kink around Tyr10 and a turn region of residues 21±25. Overall, Aβ1±40 and Aβ1±42 fibrils
show a high structural polymorphism.
Two key regions, the central residues 16±22 (β1) and the C-terminal residues 30±36 (β2),
favor β-sheet formation and promote the assembly of Aβ to form higher-order oligomers,
which can also coassemble antiparallel to form β-hairpin-rich oligomers [14±16]. Shorter Aβ
fragments are also observed to aggregate in brain tissue [
], and it provides an ideal model to
characterize Aβ oligomerizaion and pathology. Thus, truncated Aβ fragments have been
synthesized and studied, such as Aβ16±22 [
], Aβ10±35 [
] and Aβ25±35 [
] peptides. The
fragment of Aβ16±22, comprising β1-strand of the fiber models, has been extensively investigated
by experiments and computational simulations. NMR study by Balbach et al. showed that the
fragment of Aβ16±22 could form highly ordered fibrils with an antiparallel organization of
βsheets, similar to those by full-length Aβ [
]. Spectroscopic study by Lu et al. reported that the
termini-capped Aβ16±22 peptides assembled into fibers and nanotubes at neutral pH [
Performing atomistic replica exchange molecular dynamics (REMD) simulations, Gnanakaran
et al. reported that Aβ16±22 could form stable dimers aligned in parallel, antiparallel, or cross
]. Using all-atom MD simulations with explicit solvent, Nguyen et al. monitored
the growth of Aβ16±22 oligomer by adding a disordered monomer to an ordered oligomer, and
they found the dynamics of oligomer formation follows a two phase dock-lock mechanism
]. These studies have provided important information on the structural and biophysical
information of small Aβ16±22 oligomers. Nevertheless, the knowing of the Aβ30±36 fragment
that comprises β2-strand, remains elusive.
The microcrystal structure of Aβ30±35 segment was identified by Colletier et al. through its
X-ray fiber-diffraction pattern [
]. They found the fibrillar structure displays one type of
parallel β-sheet with a face-to-back steric zipper, which forms a knobs-into-holes type of packing.
Thereafter, a β-sheet amyloid mimic (BAM) derived from Aβ30±36 was crystallized [
recognized to form out-of-register fibrils or cylindrin-like, out-of-register oligomers, which
bond face to face [
]. Both types of BAM (Aβ30±36) are toxic to mammalian cells, whereas
the in-register peptide fibrils formed by Aβ30±36 show little toxicity. The contrast of peptide
interfaces reflects the structural polymorphism of Aβ30±36 oligomers, and needs to be further
clarified. The unnatural amino acid Hao applied in BAM (Aβ30±36) blocks uncontrolled
intermolecular hydrogen-bonding and promotes β-sheet formation. Therefore, the pure Aβ30±36
oligomers are supposed to have a more complex hydrogen-bonding network with respect to
BAM (Aβ30±36); meanwhile, the dominant interaction of Aβ30±36 peptide assembling also
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needs further investigating. Here we carried out replica-exchange molecular dynamics
(REMD) simulations in explicit solvent to characterize the atomic structure of capped Aβ30±36
hexamers and examine the key driving force for the oligomerization of Aβ30±36 peptides. We
also studied two mutations of Aβ30±36 (G33V and L34T) implicated in the stability of the wild
type (WT), and compared their assembling behaviors with the WT peptides. It might provide
a potential explanation of the reduced neurotoxicity induced by G33V/L34T Aβ peptides.
Materials and methods
Six Aβ30±36 (30AIIGLMV36) peptides were placed randomly in a simulation box (5.6nm×
5.6nm×5.6nm), and then underwent a 5-ns simulation at 500 K to make the residues in the
state of coil. The peptides are at neural pH with the N- and C-terminus in capped state, and
counterions are added to neutralize the system and mimic the experimental condition. We
choose Aβ30±36 hexamer for two reasons. First, Liu et al. found that β-sheet amyloid mimic
(BAM) Aβ30±36 may form cylindrin-like tetramer using X-ray crystallography [
hydrogen bond network of BAM(Aβ30±36) is similar to that of two-stranded antiparallel β-sheets,
while it has a strong interface and a weak interface. Since the pure Aβ30±36 can provide more
hydrogen bonding sites, the peptide number essential to form Aβ30±36 β-barrel should be close
to and less than eight. Second, previous Monte Carlo [
] and REMD [
] studies showed that
a β-barrel can be formed by 6~8 Aβ16±22 peptides. As Aβ30±36 peptide has the same amino acid
length as Aβ16±22, it probably needs no more than eight chains to form a stable closed β-barrel.
Since the hexamer system needs the least computational resource, we adopt Aβ30±36 hexamer
for REMD simulations.
Several mutations located at the Aβ30±36 fragment have been studied, such as Piedmont
mutant L34V, Iowa mutation G33N, etc. The L34V mutant shows a similar hemorrhagic
phenotype and elicits an analogous cell-death pathway as the WT Aβ1±40 peptide [
]. It also leads
to the aggregation and deposition of Aβ1±42 in the brain and causes the Piedmont type of
hereditary cerebral amyloid angiopathy [
]. Among these sing-point substitutions, two
mutations G33V and L34T attract our attention, because they were reported to reduce Aβ toxicity
[32±36]. Kanski et al. investigated the effect of G33V on Aβ1±42-induced oxidative stress and
neurotoxicity in cultured hippocampal neuronal cells, which shows that G33V mutant
peptides only oxidize neuronal proteins to a small extent, and cause no significant cell death [
Lecomte group studied the structures of Aβ1±42 and its L34T mutant, and found the mutants
are harmless, which may be attributed to incapacity for L34T mutants to form anti-parallel
β-sheet fibrils [
]. The systems of G33V mutant (ACE-AIIVLMV-NH2) and L34T
(ACEAIIGTMV-NH2) mutant peptides were pre-simulated at 500 K for 5 ns as the WT system
did. The obtained structures were used to as the initial states for REMD simulations (shown in
REMD simulations in explicit water
Atomistic MD simulations are performed in the isothermal-isobaric (NPT) ensemble using
the GROMACS 4.5.3 software [
]. GROMOS96 force field [
] has been widely applied to
model Aβ peptides in plenty of computational studies [24,39±42], and it can obtain Aβ
conformational propensities in agreement with NMR results [
]. Previous MD/REMD simulations
show that GROMOS96 force field is suitable for studying the Aβ aggregation, as well as the
interaction between Aβ and nanoparticles, small molecules, etc. Therefore, we select
GROMOS96 force field to model Aβ30±36 fragments. The water molecules are modeled by SPC [
We perform three REMD simulations [45±47] to study Aβ30±36 hexamer and its G33V/L34T
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mutants, with a total simulation time of 18 μs. Each system includes 40 replicas, and one
replica is a single 150-ns MD run at specific temperature ranging from 305 K to 430 K. The time
step used in MD simulations is 2 fs, and the replica exchange is attempted every 1000 steps,
resulting in an average acceptance ratio of ~22% for each system. Peptide bonds are
constrained by the LINCS algorithm [
] and water geometries are constrained by SETTLE [
The temperature is maintained constant using velocity rescaling method [
], and the pressure
is kept at 1 bar using the isotropic Parrinello-Rahman's method [
electrostatic interaction is calculated using the PME method  with a real space cutoff of 1.0 nm,
and van der Waals interaction is calculated using a cutoff of 1.4 nm.
The Daura analysis method [
] was applied to cluster the sampled conformations using a
Cαroot mean square deviation (Cα-RMSD) cutoff of 0.35 nm. A hydrogen bond (H-bond) is
considered to be formed if the distance between donor D and acceptor A is less than 3.5 Å and the
D-H-A angle is larger than 150Ê. A β-sheet angle is defined as the angle between two
neighboring β-strands in all size of β-sheets. The twist angle of a β-sheet bilayer is averaged over all the
involved β-sheet angles (supplementary angle if the β-sheet angle is obtuse). The DSSP
] is applied to determine the secondary structure. Based on the interlayer topology,
the conformations of bilayer β-sheets are denoted by m + n, where m and n respectively
represent the m- and n-stranded β-sheets forming the bilayer. More simulation details are given in
the Supporting Information.
Results and discussion
We verified the convergence of the three REMD simulations through the comparison of the
following parameters within two different time interval using 50±100 and 100±150 ns data for
WT, G33V and L34T systems, respectively: (1) probability density function (PDF) of
end-toend distance for all peptides, number of H-bonds, radius of gyration (RG), and solvent
accessible surface area (SASA); (2) the average population of different secondary structure contents
(Supporting information). As shown in S2 Fig, the distributions of the reaction coordinates
mentioned above within two independent time intervals overlap very well for all the systems.
The secondary structure contents for three systems are also quite similar within two
independent time intervals, shown in S3 Fig. These results suggest that our REMD simulations are
WT, G33V and L34T Aβ30±36 peptides in the oligomers display
differences in secondary and tertiary structures
To examine the structural properties of WT, G33V and L34T aggregates at 310K, we calculated
the dominant secondary structure (coil and β-sheet) probability of each residue in Fig 1. It
shows that Aβ30±36 hexamer has an average β-sheet content of 60.1% in all at 310 K, and the
residues located at the middle of the WT Aβ30±36 sequence have a higher probability to adopt
β-sheet conformations than those at the ends. The inclination of Aβ30±36 oligomers for β-sheet
formation is in good agreement with experimental observations [
]. After the
substitution of G33 by V, the average coil propensity is slightly increased (from 38.4% to 41.3%) and
the β-sheet propensity reduced, in which M35 contributes the most. Interestingly, previous
studies suggest that G33 is a possible site of free radical propagation processes that are initiated
on M35, which is involved in Aβ toxicity [
]. Although our model cannot simulate the
proton transfer process, the results still reflect that G33V mutation influences the structure of
M35 the most among all the residues. Meanwhile, nearly all the residues of the L34T mutants
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Fig 1. Propensity of dominant secondary structure contents for each amino acid residue in wild type
(WT), G33V and L34T Aβ30±36 hexamers at 310K: coil propensity (a) and β-sheet propensity (b). Error
bars were calculated using the 50±100 ns and 100±150 ns data.
show an increment in coil propensity and a reduction in β-sheet propensity. The difference of
secondary structures between the WT peptides and mutants reflects that the substitution of
L34 by T has a significant prevention of the β-sheet formation of Aβ30±36 hexamer. Recent
spectroscopic studies show that the L34T mutation alters the oligomeric structure and
prolongs the lag phase of Aβ1±42 fibrillation, accompanied with decreased toxicity [34±36]. The
βsheet reduction of Aβ30±36 segments duo to the L34T substitution, is supposed to go against
the fiber formation of full-length Aβ1±42 and the oligomerization at the early stage. These
results indicate that the WT Aβ30±36 hexamer prefers to form β-sheet, and the G33V and L34T
mutations have a different influence on the secondary structure of Aβ30±36 assemblies.
The temperature dependence of secondary structures for WT, G33V and L34T Aβ30±36
hexamers was examined by calculating their average coil and β-sheet propensities, shown in
Fig 2. As the temperature increases, the WT Aβ30±36 hexamers display a gradual rising coil
Fig 2. Average coil (a) and β-sheet (b) propensities of WT, G33V and L34T Aβ30±36 hexamers at
different temperatures. The error bars were calculated using the 50±70 ns, 70±90 ns, 90±110 ns, 110±130
ns, and 130±150 ns data.
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propensity, with 36.4±0.3% at the lowest temperature of 305 K and 52.5±1.2% at the highest
temperature of 430 K, and a gradual decline of β-sheet propensity, with 62.5±0.3% at 305 K
and 40.9±1.5% at 430 K. This indicates that the WT Aβ30±36 peptides prefer to aggregate at
lower temperatures, consistent with other amyloid sequences, such as p53
aggregation-nucleating 251ILTIITL257 [
], Aβ16±22 [
] and Aβ29±42 peptides [
]. As for G33V mutants, they
have similar temperature dependence as WT, with a β-sheet propensity of 58.8±1.0% at 305 K
and 51.6±2.3% at 430 K. With respect to WT hexamers, the G33V mutants have a lower
βsheet propensity in the range of 305±383 K and a higher β-sheet propensity in the range of
387±430 K. As for L34T mutants, they have a significantly lower β-sheet propensity than WT
peptides at all the simulated temperatures, with a propensity of 48.0±1.6% at 305 K and 13.6
±1.1% at 430 K.
We then performed a RMSD-based cluster analysis for 50000 conformations sampled for
each system at 310K using the procedure described in the methods subsection. Using a
CαRMSD cutoff of 0.35 nm, the conformations of Aβ30±36 WT and its G33V/L34T mutant
oligomers are separated into 139, 112 and 266 clusters, respectively. Fig 3 shows the central structure
and corresponding population of the first eight most-populated clusters. These clusters
represent 43.4%, 44.6%, and 37.3% of all conformations of wild type, G33V and L34T hexamers,
respectively. The Aβ30±36 WT hexamers primarily adopt ordered β-sheet-rich conformations,
comprised of β-barrel, 4 + 2 and 3 + 3 β-sheet bilayer, in good agreement with the X-ray study
in which the crystallographically determined BAM Aβ30±36 can form cytotoxic β-barrel-like
oligomers and fibrils [
]. A closed β-barrel hexamer was observed in Cluster-1, with the
βstrands in out-of-register alignments. Conventional MD simulations initiated from Cluster-1
Fig 3. Representative conformations of the first eight most-populated clusters for WT (a), G33V mutant (b), and L34T mutant (c)
Aβ30±36 hexamers at 310K. The corresponding population of each cluster is given in the parentheses.
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display a small RMSD (<0.2 nm) as a function of time (see S8 Fig), revealing that the β-barrel
structure is quite stable. Similar β-barrels composed of Aβ16±22, Aβ25±35 or K11V display a stable
existence in the previous computational or experimental studies [
], suggesting that
the cylindrin-like oligomer is probably an on-pathway intermediate involved in out-of-register
amyloid fibril formation . These cylindrins and β-barrels can reduce cell viability via a
proposed mechanism of interacting with membranes [61±63]. Through conventional MD
simulations, the 4 + 2 β-sheet bilayer is observed to be able to transfer to a closed barrel-like structure
(see S4 Fig), accompanied with enlarged structural fluctuation and a β-sheet-coil transformation
when the H-bonds are destructed. The β-strands in one bilayer are orthogonal to those in the
other bilayer at first, and the two-stranded bilayer can drift away from the four-stranded bilayer.
The structural flexibility of the 4 + 2 β-sheet bilayer in Cluster-2 implies that it is probably an
intermediate in touch with β-barrel and 3 + 3 β-sheet bilayer. Similar structural flexibility of
fibril-barrel transitions has recently been observed by Zhang et al. in a
replica-exchange-withtunneling simulation study of three K11V peptides [
]. They found a key transition state
connecting the fibril and cylindrin pathways, in which peptides have not yet associated by
Compared with the conformations of Aβ30±36 WT oligomers, those of G33V mutant have a
higher similarity and predominantly adopt β-sheet-rich bilayers. Among the first eight
mostpopulated clusters, 4 + 2 β-sheet bilayer is observed in Cluster-3, and 3 + 3 β-sheet-rich bilayers
in all the other clusters. We also observed well-formed β-sheet twists in the 3 + 3 β-sheet
bilayers, whose twist angles range from 12 to 32 degree. Our result is larger than the twist angle
obtained from the well-formed fiber structure [18,65±67]; because the simulated hexamer has
a much smaller size than the mature fiber, and it is easily interfered by water molecules. The
inter-mainchain H-bonding map of the first eight most-populated clusters in S5 Fig shows
that the G33V Aβ30±36 peptides can adopt in-register parallel (major) or antiparallel (minor)
β-sheets, and two-residue-shift out-of-register parallel (medium) β-sheets, evolutionarily more
optimized by β-sheet twists. Similar twisted morphologies of antiparallel β-sheet have been
reported in the study of membrane-bound Aβ pore and β2m83±89 oligomers [
]. On the
contrary, the L34T mutants display a higher diversity and have more β-bridge (tan-colored
parts in Clusters-2, 3, 5 and 8) as well as bend (cyan-colored parts) content. The disordered
structures are hard to classify according to their tertiary topology, and small hairpin loop and
hairpin are observed in Clusters-1 and 3, respectively. The ACE group, residues I31, T34 and
V36 of one peptide in Cluster-1 is observed to form H-bonds with the closest peptides, as
shown in S6 Fig. This hairpin loop peptide helps to stabilize the oligomeric structure through
H-bonding with the neighbor peptides. One peptide in Cluster-3 displays a hairpin
conformation with two pairs of intrachain H-bonds (I32-NH2 and I32-M35, shown in S6 Fig). This
hairpin peptide also forms H-bonds with intralayer strand, but does not form H-bonds with
In order to investigate the dominant secondary structure (coil and β-sheet) probability of the
most-populated conformations, we calculated the coil and β-sheet propensity as a function of
cluster index for each system at 310K in Fig 4. It shows that the WT and G33V/L34T Aβ30±36
hexamers display a different secondary structure content distribution. Using Daura cluster
analysis method, the cluster with lower index has a higher sampling probability. Although the
substitution of G33 by V has a slight influence on the total population of secondary structure, it
remarkably increases the coil component in the first eight most-populated conformations,
despite of a slight decrease of 1.1% for Cluster-3 and 0.2% for Cluster-6; it also reduces the
βsheet component, despite of a slight increase of 3.6% for Cluster-3 and 0.7% for Cluster-6. This
indicates G33V mutant has a lower probability to form β-sheet-rich structures with respect to
WT Aβ30±36. The difference of WT and G33V mutant peptides in secondary structure may
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Fig 4. Averaged propensity of coil (a) and β-sheet (b) as a function of cluster index in WT, G33V and
L34T Aβ30±36 hexamers at 310K.
result from the change of backbone dihedral angle distribution, and the peptides still favor to
form β-sheet (see S7 Fig). It is reminiscent of a previous computational study, in which Lu et al.
applied the coarse-grained protein OPEP force field to investigate the effects of G33I mutation
on the structures of Aβ29±42 monomer and dimer. Interestingly, they found the G33I mutants
are more disordered than WT dimer, and display less β-sheet content in the C-terminal
residues, with a slightly increased population of parallel alignments [
]. As for the substitution of
L34 by T, it not only reduces the β-sheet content of Aβ30±36 hexamer, but also significantly
reduces the probability to form β-sheet-rich structures. These indicate that both G33V and
L34T mutant Aβ30±36 hexamers have lower β-sheet content than WT peptides in the
WT Aβ30±36 peptides mainly assemble into β-barrel or β-sheet-rich
oligomers, while G33V substitution leads to more extended β-sheet-rich
hexamers, and the L34T mutants have more complex H-bonding
To have an overall view of the conformational distribution of WT and G33V/L34T mutant
Aβ30±36 oligomers, we plotted the two-dimensional free energy landscape (or potential of
mean force, PMF) as a function of H-bond number and RG in Fig 5. As shown in Fig 5A,
the free energy surface of WT Aβ30±36 oligomers is broad and the minimum-energy basin is
shallow, with the number of H-bonds ranging from 12 to 35 and the RG ranging from 0.85
to 1.1 nm. Considering the centers of the most-populated conformations (including
β-barrel, 4 + 2 and 3 + 3 β-sheet bilayer) projected in this H-bond number-RG plane are close,
twenty-four 100-ns independent conventional MD simulations were performed to check if
these structures can convert to each other. The low converting ratio (one out of 24 runs, see
S8 Fig) indicates that the transformation is energetically very costly, suggesting that the
oligomeric structures might be involved in distinct aggregation pathways of Aβ30±36 peptides.
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Fig 5. Free energy landscape (in kcal/mol) for WT (a), G33V mutant (b), and L34T mutant (c) Aβ30±36
hexamers at 310 K, projected in the two-dimensional plane of the intermolecular H-bond number and
radius of gyration (RG). Cluster numbers corresponding to the representative structures of three systems in
Fig 3 are respectively marked in the plane.
Especially for the intralayer rearrangement of strand alignment, a complete H-bonding
network needs reforming.
After the substitution of G33 by V, the intermolecular H-bond number of Aβ30±36 hexamers
ranges from 15 to 33, and the RG ranges from 0.90 to 1.15 nm. The increased RG reflects that
the β-barrel structures are distinctly reduced and more residues are exposed to water solution.
The hydrophobicity of valine makes the peptides less compacted, which implies the geometric
occupation of its sidechain disturbs the packing of residue sidechain and as a result changes
the oligomeric morphology. As for the L34T Aβ30±36 oligomers, the H-bond number ranges
from 9 to 35, and the RG ranges from 0.85 to 1.08 nm. The more disperse distribution of
Hbond number indicates that the L34T hexamers have reduced β-sheet content and more
Distributions of β-sheet angle of WT and G33V/L34T mutant Aβ30±36 oligomers are
given in Fig 6A to quantify the parallel and anti-parallel β-sheet frequency. It shows that the
β-sheets formed by WT Aβ30±36 peptides have a slight preference for parallel orientation,
with a parallel/anti-parallel percentage of 61.6%/38.4%; G33V β-sheets significantly tend to
be parallel aligned; L34T β-sheets have a preference of out-register antiparallel alignment.
The end-to-end distance probability distributions for all chains are presented in Fig 6B.
There is a sharp peak located at 1.8 nm in WT and G33V peptide systems, corresponding to
β-strand or β-sheet-rich conformations. The structural ensemble of L34T peptides gets
broader, with the highest peak located at 1.8 nm and two smaller peaks at 0.6 nm and 1.1
nm (see S2A Fig for clarity), respectively. The PDF peak of 0.6 nm for a L34T mutant
corresponds to the hairpin structure, and the peak of 1.1 nm corresponds to the meander
structure, namely hairpin loop (shown in S6 Fig). These indicate that L34T peptide in the
oligomers is less extended than WT peptide, corresponding to less β-sheet content. Fig 6C
and 6D show the probability distribution of SASA and the contribution to SASA per
residue, respectively. The G33V mutant oligomers display an increased surface area exposed to
water than the WT Aβ30±36, attributed to the hydrophobic sidechain of valine. As for the
L34T mutant oligomers, the SASA of A30, I32, L34 and V36 is increased, while the SASA of
I31, G33 and M35 is reduced. The adjacent switch of SASA increment reveals that the
I31-G33-M35 face of the Aβ30±36 peptide prefers to orientate to the interior of oligomers
after the L34T substitution. Note that if the SASA is normalized by the total surface area of
all chains (see S9 Fig), the enhanced hydrophobicity induced by the G33V mutation indeed
decreases the normalized SASA, and the distribution of normalized SASA for L34T mutant
oligomers remain the same as the WT peptides.
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Fig 6. Analyses of peptide β-sheet angle, end-to-end distance, and solvent accessible surface area
(SASA) for WT, G33V mutant, and L34T mutant Aβ30±36 hexamers at 310 K: (a) probability density
function (PDF) of β-sheet angle; (b) PDF of end-to-end distance for all peptides; (c) PDF of SASA for all
residues; (d) SASA as a function of residue index.
WT and G33V oligomers are mostly stabilized by hydrophobic
interaction, while L34T oligomers are stabilized by both hydrophobic and
To probe the primary peptide-peptide interactions disturbed by the mutations and the key
residues for β-sheet formation, we plotted in Fig 7 the mainchain-mainchain (MC-MC) and
sidechain-sidechain (SC-SC) contact probability map between all residue pairs of WT, G33V
mutant, and L34T mutant Aβ30±36 oligomer, respectively. The relatively smooth MC-MC
contact probabilities of WT peptides reveal that the capped Aβ30±36 peptides assemble with no
preferred orientation. The I32-I32, I32-L34 and L34-L34 pairs have a high SC-SC contact
probability of 15.8%, 19.4%, and 26.5%, respectively, indicating the hydrophobic interaction
plays an important role in peptide-peptide interplay. This is consistent with a recent NMR
study of macrocyclic Aβ30±36 tetramer, in model of which the sidechains of I32 and L34 have a
strong packing [
When G33 is substituted by V, the peptides present weakened MC-MC contacts and have a
tendency to be parallel aligned. The SC-SC contacts show the interaction between the sidechains
of I32 and L34 are greatly lessened, and those of I31-I32 and V33-L34 pairs are enhanced. It
reveals that the increased hydrophobicity brought by G33V mutation alters the associations
between other residues and also interferes with the MC-MC interaction. Too much hydrophobic
interaction is reported to have a negative effect on the protein stability as well as the formation of
an aggregative nucleus in peptides-hydrophobic surface system [
], or to reduce the β-sheet
content of fibrils and lead to disordered oligomers in Aβ16±22-crowder system [
]. In a previous
REMD simulation study of G33I mutant Aβ29±42 dimer, the enhanced hydrophobic at G33 was
also reported to reduce intermolecular interaction in WT dimer [
]. Our REMD results show
that the hydrophobic sidechain of V33 disrupts the β-barrel and increase coil content in
mostpopulated clusters of Aβ30±36 assemblies. After the substitution of L34 by T, the MC-MC contact
map displays a much higher probability along the left diagonal, indicating that the L34T mutant
has a preference of out-register antiparallel alignment and the C-terminal residues tend to be
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Fig 7. The interpeptide mainchain-mainchain (MC-MC) (a) and sidechain-sidechain (SC-SC) (b) contact probability maps of
REMD-generated conformations of WT, G33V mutant, and L34T mutant Aβ30±36 oligomer at 310 K.
oriented to water environment. This agrees with the structural characterization of less toxic Aβ
mutant (L34T) fibrils using tip-enhanced Raman spectroscopy, in which the mutants align slightly
less parallel and more antiparallel compared with WT peptides [
]. For SC-SC interactions, the
interplays between I31 and M35 (I31-I31, I31-M35, and M35-M35 pairs) have the highest contact
probabilities in replace of those between I32 and L34, indicating the L34T substitution makes the
sidechain of I31 and M35 prefer to be buried in the interior of oligomers, which is consistent with
the SASA analyses in Fig 6. These are attributed to the hydrogen bonding between T34 and other
chains and the increased hydrophilicity of C-terminal residues brought by L34T mutation.
To clarify the difference of peptide interaction as a result of the G33V substitution, we
presented the interpeptide MC-MC and SC-SC contact probability between I32/L34 and other
residues in Fig 8. The MC-MC contacts become less in all; the SC-SC contact probabilities of
I32 and L34 get smaller after the G33V substitution, whereas those of I31 and M35 become
higher (except for a decrease of 2.3% for I32-M35 pair that may be involved in other sidechain
packing). According to the X-ray crystallographic observation [
], the cylindrin-like or
fibrillar BAM (Aβ30±36) oligomer prefers to bond face to face, with the sidechains of I32 and L34
buried in interior, which is consistent with the highest SC-SC contact probability of I32 and
L34 for WT peptides. After the substitution of G33 by V, the I31-V33-M35 face of peptide has
an increment of hydrophobicity, and it competes with the I32-L34 face on sidechain packing.
The steric-zipper effect between the I31-V33-M35 and I32-L34 face also makes the peptides
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Fig 8. The interpeptide MC-MC (a) and SC-SC (b) contact probability between I32/L34 and other
residues for WT and G33V system at 310 K. The blue and orange columns correspond to I32 and L34,
respectively; the blank and filled columns correspond to WT and G33V Aβ30±36 oligomers, respectively.
tend to bond face to back. G33I is another mutation identified in Aβ30±36 region, with a similar
physical and chemical nature. Harmeier et al. reported that this mutation promotes the
aggregation process of Aβ1±42 by forming a continuous hydrophobic surface, and makes peptides
more easily to form higher oligomers, which leads to less toxicity [
]. Reminiscently, the
G33V substitution of Aβ30±36 peptides in our simulations makes the I31-V33-M35 face a
continuous hydrophobic surface, which competes with the I32-L34 face in sidechain packing. As a
result, the dry interior formed by I32 and L34 is disrupted, and the cylindrin-like Aβ30±36
oligomers are significantly reduced and the fibrillar oligomers increase.
We further examined the perturbation of the L34T substitution on the residue-based
peptide-peptide interaction in the term of number of H-bonds, and plotted the intermolecular
Hbond distribution in Fig 9A. It shows that the L34T oligomer form 1~2 less MC-MC hydrogen
bonds and 1~2 more MC-SC hydrogen bonds than the WT peptides. The shift in the H-bond
number is attributed to the formation of H-bonds between the hydroxyl groups of T34
sidechain and the backbone of Aβ30±36 peptides. As shown in Fig 9B, the sidechains of T34 favor
the polar threonine with hydrogen bonding, and have no preference to the other hydrophobic
residues. Fig 9C displays the snapshots of MC-SC H-bond formed in the conformations of
Cluster-3 and Cluster-6, respectively. Our calculation and detailed structural check show that
there is no sulfur atom involved in hydrogen bonding under the criterion mentioned in
Methods section. It has been demonstrated that interpeptide hydrogen bonding and hydrophobic
interaction play important roles in the formation and stabilization of Aβ aggregates [23,73±
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Fig 9. Analyses of the intermolecular H-bond distribution for WT and L34T Aβ30±36 oligomer at 310 K:
(a) the PDF of MC-MC and MC-SC H-bond number; (b) the average number of H-bonds formed
between T34 sidechain and individual residue mainchain; (c) a local glance upon the conformations
of Cluster-3 and Cluster-6. The names of the residues involved in MC-SC H-bonding are highlighted in
orange. The protein is represented in licorice, the secondary structure in cartoon, and the MC-SC H-bond in
orange dashed line. Carbon atoms are colored in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur
atoms in yellow, and hydrogen atoms in white, respectively, in the snapshots.
75]. Given the inability of WT Aβ30±36 sidechains to form H-bonds, the hydrogen bonding
between sidechains of T34 and mainchains of other peptides disarranges the interpeptide
H-bond network and leads to disordered oligomers.
Using REMD simulations, we have investigated the hexameric structures of termini-capped
Aβ30±36 peptides and examined the effect of G33V/L34T mutations on the oligomeric
assemblies. Our results revealed that the Aβ30±36 hexamer has an average β-sheet content of 60.1%
and mainly adopts conformations of β-barrels, 4 + 2 and 3 + 3 β-sheet bilayers at 310 K. The
strands in β-barrels display out-of-register alignments; bilayer β-sheets include parallel,
orthogonal, and antiparallel ones. The hydrophobic interaction between I32 and L34 residues
plays a critical role in the assembly and structural stability of Aβ30±36 hexamers. The G33V
mutants show less β-sheet contents in the most-populated (top 44.6%) conformations, and
adopt β-sheet-rich bilayers on the whole, with strands mainly in in-register parallel alignments.
The I31-V33-M35 face of peptides tends to orient to the I32-L34 face, and their steric-zipper
effect interferes with the face-to-face sidechain packing of I32-L34 face in WT Aβ30±36. The
L34T mutants have a significant β-sheet reduction and a higher structural diversity, including
quite a few disordered and hairpin-like oligomers. It is mainly attributed to the hydrogen
bonding of T34 sidechain with peptide backbones, which disturbs the intermolecular H-bond
network in WT Aβ30±36. Overall, both the G33V and L34T mutations disrupt the Aβ30±36
βbarrel conformations that are closely related to cell toxicity, and weaken the I32-L34
hydrophobic interaction. Our REMD results provide structural insights into the assembly of WT
and G33V/L34T mutant Aβ30±36 peptides, which is helpful for the development of
amyloidbased nanostructures and the design of novel inhibitors.
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S1 Text. Supplementary simulation details.
S1 Fig. The initial structures of WT (a), G33V (b) and L34T (c) Aβ30±36 hexamer for
REMD simulation. The peptides are in cartoon representation.
S2 Fig. The probability density function (PDF) of end-to-end distance for all chains (a),
number of H-bonds (b), radius of gyration (RG) (c), and solvent accessible surface area
(SASA) (d) for three systems within two independent time intervals of 50±100 ns and 100±
150 ns at 310K.
S3 Fig. Probability of secondary structures within two independent time intervals of 50±
100 ns and 100±150 ns for WT, G33V and L34T Aβ30±36 hexamer systems at 310K.
S4 Fig. Cα-root mean square deviation (Cα-RMSD) of WT Aβ30±36 hexamer as a function of
time, with corresponsive secondary and tertiary structures. Both conventional MD simulations
are initiated from the 4 + 2 β-sheet bilayer in Cluster-2: (a) the peptides transfer to a closed
barrellike structure; (b) the two-stranded bilayer drifts away from the four-stranded bilayer.
S5 Fig. The interpepide H-bonding map between backbones of the first eight
most-populated conformations for WT, G33V and L34T Aβ30±36 hexamer systems. The color indicates
the average number of H-bonds. AP0/P0 represents in-register antiparallel/parallel β-sheets;
P1/P2 represents 1-residue-shift/2-residue-shift out-of-register parallel β-sheets.
S6 Fig. Snapshots of one L34T mutant Aβ30±36 peptide with an end-to-end distance of 5.77
Å (a), 9.66 Å (b) and 17.90 Å (c). The pane-contained H-bonds are highlighted in orange,
with explicit names of the residues involved in H-bonding. The end-to-end distance is
calculated from the A30 Cα atom to the V36 Cα atom. The secondary structures are shown in
cartoon representation, and the peptides in licorice representation with carbon atoms in cyan,
oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow and hydrogen atoms in
S7 Fig. The distribution of dihedral angles of the first eight most-populated
conformations for WT, G33V and L34T Aβ30±36 hexamers at 310K: (a) the probability in dihedral
angle φ-ψ plane; (b) PDF of φ and ψ.
S8 Fig. The time evolution of Cα-RMSD for WT Aβ30±36 hexamer. These conventional MD
simulations are initiated from the conformations in the first six most-populated clusters.
Different colors represent independent MD runs. The green line of Cluster-2 corresponds to a
transformation from a 4 + 2 β-sheet bilayer to a closed barrel-like structure.
S9 Fig. The PDF of SASA which is normalized by the total surface area of all chains for
WT, G33V and L34T Aβ30±36 hexamer systems at 310K.
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Simulations were performed at the High Performance Computing Server of Shanghai
University of Sport. We thank Dr. Guanghong Wei for helpful discussion.
Data curation: Zhenyu Qian, Qingwen Zhang.
Formal analysis: Zhenyu Qian, Yu Liu, Peijie Chen.
Investigation: Qingwen Zhang.
Writing ± original draft: Zhenyu Qian, Qingwen Zhang, Peijie Chen.
Writing ± review & editing: Zhenyu Qian, Yu Liu, Peijie Chen.
15 / 18
16 / 18
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