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Journal of Alzheimer’s Disease 47 (2015) 215–229 215

DOI 10.3233/JAD-150046
IOS Press

Modeling the Aggregation Propensity and

Toxicity of Amyloid-␤ Variants
Manish K. Tiwari and Kasper P. Kepp∗
Technical University of Denmark, DTU Chemistry, Kongens Lyngby, Denmark

Handling Associate Editor: George Acquaah-Mensah

Accepted 11 April 2015

Abstract. Protein aggregation is a hallmark of many neurodegenerative disorders. Alzheimer’s disease (AD) is directly linked
to deposits of amyloid-␤ (A␤) derived from the amyloid-␤ protein precursor (A␤PP), and multiple experimental studies have
investigated the aggregation behavior of these amyloids. The present paper reports modeling of the aggregation propensities and
cell toxicities of genetic variants of A␤ known to increase disease risk. From correlation to experimental data, and using four
distinct experimental structures to test structural sensitivity, we find that the Spatial Aggregation Propensity (SAP) formalism
can describe the relative experimental aggregation propensities of A␤42 variants (R2 = 0.49 and 0.70, p ∼ 0.02 and 0.002, for
1IYT and 1Z0Q conformations using a probe radius of 10 Å). Our analysis finds correlation between the reduction in hydrophilic
surface and experimental aggregation propensities. Finally, we show that experimental cell toxicities of A␤ variants are well
described by computed SAP values, suggesting direct interplay between aggregation propensity and cell toxicity and providing
a step toward a first computational estimator of A␤ toxicity. The present study contributes to our understanding of amyloid
aggregation and suggests a method to predict aggregation propensity and toxicity of A␤ variants, and potentially to reduce
aggregation propensities of amyloids by molecular intervention directed toward specific conformations of the peptides.

Keywords: Alzheimer’s disease, amyloid-␤, hydrophilic surface, protein aggregation, structure-activity relations

INTRODUCTION AD is recognized as a major epidemic and one

of the major challenges of the current century [10]
During protein aggregation, protein parts or pep- with steadily increasing prevalence [10, 11]. Extracel-
tides self-assemble to produce small soluble oligomers lular aggregates of amyloid-␤ (A␤) peptides, “senile
or fibrillar aggregates, typically cross-linked ␤-sheets plaques”, are a primary pathological hallmark of AD
[1–4]. As a central characteristic of many human dis- [12, 13]: These peptides are cleaved from the amyloid-
eases such as Alzheimer’s disease (AD), Parkinson’s ␤ protein precursor (A␤PP) found in membranes of
disease, and Huntington’s disease, prion diseases, and cells and intracellular organelles [14, 15]. The plaques
type II diabetes [2, 3, 5], research into aggregation consist of A␤ isoforms of variable length, primarily
processes have attracted substantial attention [5–8]. A␤40 and A␤42 , that are post-translationally modified
Pathological conditions are thought to emerge from by oxidation and metal binding [11, 16].
conformational changes in the normal, native states AD is predominantly sporadic in nature, i.e., with
of the peptides that lead to gain-of-toxic-function or family history seen in only a small minority of cases
loss-of-normal-function [5, 9]. [2, 11] with multiple contributing risk factors from
genes, environment, and lifestyle [17, 18]. However,
∗ Correspondence to: Kasper Planeta Kepp, Department of Chem-
genetic variations are known to give rise to some
istry, Technical University of Denmark, DK 2800 Kongens Lyngby,
forms of early-onset familial AD (FAD) [19]. These
Denmark. Tel.: +45 45 25 24 09; Fax: +45 45 88 17 99; E-mail: variations are found mainly in A␤PP [20, 21] and in presenilin (PSEN) [22, 23], which is the catalytic unit

ISSN 1387-2877/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
216 M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids

of the ␥-secretase enzyme complex that produces A␤ that are known clinically to increase risk of disease. We
upon proteolytic cleavage of A␤PP [24, 25]. Due to correlated computed and experimentally determined
this genetic evidence and the amyloid deposits found aggregation propensities, using several distinct A␤
in brains of patients, A␤ is acknowledged as having a NMR structures that represent a spectrum of structures
central role in the disease [26–28]. relevant to in vivo conditions. Computation of protein
It is now known that soluble oligomers of A␤ are the aggregation in relation to neurodegenerative disorders
more toxic species [29–31]. This toxicity can be aggra- has been a long-standing research goal [61]. However,
vated by chemical aging processes [32]. Furthermore, as far as we know, we present here the first study that
interactions with cell membranes during aggregation systematically compares computed and experimental
are important in modulating A␤ toxicity [33–36]. A aggregation propensities of known genetic A␤ variants
two-step membrane disruption mechanism was identi- related to disease.
fied recently [34–36]. First, ion-selective channels are Our findings provide new structural and chemical
formed, then disruption and fragmentation of the mem- detail to amyloid aggregation, suggest central struc-
brane occurs during fibril formation, a process that is tural features that serve as likely precursors, and show
aggravated by gangliosides [34]. Membrane disruption options for future computational estimation of aggre-
has also been reported for other proteins such as islet gation propensities and toxicities of amyloid variants.
amyloid polypeptide [37, 38]. The structures and properties identified as important
Among the isoforms of A␤ [39], the longer tend to for aggregation and toxicity may also potentially be tar-
be more hydrophobic and thus more prone to aggre- geted in rational design of amyloid-modifying drugs.
gation [2, 40], and of the two major isoforms, A␤42
(typically 10% of the total amyloid load, but the major MATERIALS AND METHODS
form in deposited plaques) is more cytotoxic than the
A␤40 isoform [41]. Consistent with this, several A␤PP Structural models of amyloids
mutations likely disturb ␥-secretase activity to increase
the A␤42 /A␤40 ratio [42]. Despite these advances, the Monomers of A␤ are all mixtures of short helices
activeconformationsandchemicalpropertiesofthenor- and disordered regions, in contrast to insoluble extra-
mal A␤ monomers that serve as precursors in oligomer cellular fibrils formed upon aggregation, which are
formation are currently unknown and hard to study largely of ␤-sheet character [62, 63]. We have consid-
experimentally due to their intrinsic disorder [43]. ered four distinct experimental structures of wild-type
Although recent clinical trials have failed to avert (WT) A␤, two for each of the most abundant isoforms;
A␤-mediated pathogenesis [44, 45], new promising A␤42 : 1IYT [64], 1Z0Q [65] and A␤40 : 1BA4 [66],
strategies use molecular structural information to tar- 2LFM [67] (Fig. 1A–1D). To compare these struc-
get A␤ toxicity [46, 47]. The need to delineate the tures, we calculated the ensemble root mean square
fundamental chemical properties of A␤ is under- deviation (RMSD) of the NMR structures for A␤42
lined by the role of post-translational modifications, and A␤40 (Supplementary Tables 1–4). Compared to
notably truncations, metal ion binding, and oxida- other reported full-length (A␤42 or A␤40 ) WT apo
tions of amyloids, and the generation of reactive monomer structures (1BA6 [68], 1AML [69]), 1IYT
oxygen species [11, 47–51]. Computational studies [64], 1Z0Q [65], 1BA4 [66], 2LFM [67], the four
have been directed towards the molecular structure structures employed in this study have well-defined
and dynamics of A␤ [52], A␤ fibrils [53], protofib- conformational ensembles, with conformations within
rils [54], oligomers [55], and various polymorphisms each ensemble showing strong resemblance; this ren-
[56]. Computational efforts have also shown promise ders a structure-property analysis meaningful. The four
toward design of inhibitors of A␤ aggregation [57, 58] structures represent a spectrum of chemical environ-
and understanding of metal ion binding to A␤ [59, ment and, for the same reason, different helix character,
60]. Still, correlation between molecular properties and as seen in Fig. 1.
experimental aggregation propensities of A␤ remains
unexplored. It is thus of major interest to understand Mutant modeling
the properties and structures that determine A␤ aggre-
gation propensities and whether these propensities can Structural modeling of A␤42 and A␤40 mutants was
be accurately predicted by computations. carried out as described recently [70]. Mutant struc-
To approach these challenges, we set out to model tures were obtained by replacing side chains using the
the aggregation propensities of the genetic A␤ variants “build mutant” module [70–72] of Discovery Studio
M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids 217

Fig. 1. WT and mutant structures of A␤42 and A␤40 . A, B) The NMR coordinates of WT structures of A␤42 (PDB: 1IYT and 1Z0Q) with 15
mutant residues shown in sticks. C, D) The NMR coordinates of WT structures of A␤40 (PDB: 1BA4 and 2LFM) with 13 mutated residues.
Colors range from yellow (negative hydrophobicity) to blue (positive hydrophobicity), via green (zero hydrophobicity) (the figure was generated
using D.S. 4.0 visualizer).

4.0 (DS4.0) [73]. Briefly, ten conformations of each structure-dependent aggregation propensity for each
mutant were generated and evaluated based on con- atom using the CHARMm force field, and the total
formational energy scoring functions, to produce four aggregation propensity is obtained as the sum of its
WT and 56 mutant structures. These were subsequently atomic aggregation scores [74].
modeled as described in detail in the Supplementary Total solvent accessible surface area was computed
Material. using the default parameters of DS4.0, notably a
probe radius of 1.4 Å, and specific residue solvent
Modeling of surface properties and aggregation accessibilities were also collected. The hydrophobic
propensities and hydrophilic surfaces of each A␤ species were
determined using DS4.0 default parameters [70]. Sup-
In addition to the total solvent accessible surface area plementary Tables 9–12 show the computed total,
and the hydrophobic and hydrophilic surfaces [70], hydrophilic, and hydrophobic surfaces for all con-
we computed the spatial aggregation propensity (SAP) formations of all the mutants and WT amyloids.
for all the 60 structures. The SAP values (Supple- Decomposition into hydrophilic and hydrophobic sur-
mentary Tables 5–8) were obtained using the method faces was based on the Kyte and Doolittle scale [76]
described by Chennamsetty et al. [74] as implemented for individual residues. To provide further validation,
in DS4.0, using both R = 5 Å and R = 10 Å to test sen- we also calculated the same surface areas for the 60
sitivity to surface resolution. This method uses the structures using the POPS (Parameter OPtimised Sur-
spatial conformations of the residues and their Black faces) server [77] with probe radius 1.4 Å; the results
and Mould hydrophobicity index [75] to calculate a can be found in Supplementary Tables 9–12.
218 M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids

Experimentally derived data set for correlation Two aggregation propensities for E22G have been
studies reported by Nilsberth et al. [85] and Hori et al. [84] (0.3
and 4.4: value normalized against WT). Similarly, two
For the known genetic variants of A␤ which relate to aggregation propensities for D23N have been reported
disease, we have compiled a table of all relevant clin- by Nilsberth et al. [85] and Van Nostrand et al. [86] (0.7
ical and biochemical data on aggregation propensities and 3.0: value normalized against WT). The aggre-
[78–81] and cytotoxicities (EC50 ) [79, 80] (Supple- gation propensities reported for E22G by Hori and
mentary Table 13). This database serves a useful role in coworkers and for D23N reported by Van Nostrand and
pinpointing important chemical properties of amyloids coworkers; are consistent with the procedure and val-
but also, as done in this work, for testing property cal- ues of other data and were thus used (Supplementary
culation methods and for understanding the chemical Table 13).
origins of, e.g., aggregation propensity. In the database,
to put numbers from different experiments on the same Statistical correlation between amyloid properties
scale, we normalized WT values to 1. As shown from
the statistical correlations below, this normalization We investigated the linear regressions between the
makes the data commensurable despite heterogeneities computed properties described above, including the
in lab protocols. SAP, and relative, normalize experimental aggregation
Thioflavin T (ThT) based fluorescence is the most propensities and EC50 values for all WT and mutant A␤
common way to quantitatively estimate the aggrega- structures. The data sets used for these correlation anal-
tion of peptides. The technique does not distinguish yses are tabulated in the Supplementary Tables 14–17.
various aggregated or oligomerized forms such as pre- A comprehensive list of all the statistical values (R2 and
fibrillar and fibrillar aggregates [82]. ThT itself may p) is provided in Supplementary Tables 18–21. In the
also interact with exogenous compounds to affect the discussion, we have considered values of R2 > 0.3 and
measured ThT fluorescence [83]. Yet, this technique p < 0.05 significant for discussion, i.e., the discussed
is the one that researchers have used and thus forms relations are unlikely (<5%) to have occurred by coin-
the basis of the experimental data. However, to vali- cidental agreement of heterogeneous data but most
date the use of these data, two independent data sets of likely resemble true relationships between chemical
aggregation propensities were considered. properties of monomer seeds, aggregation propensi-
One data set was derived from the aggregation ties, and cytotoxicities.
propensities reported by Murakami et al. [79], Zhou
et al. [81], and Hori et al. [84] for A␤42 based on ThT RESULTS AND DISCUSSION
fluorescence intensities after eight hours of incubation
time. These data sets provide good numerical spread in Structures used for computation
aggregation propensities allowing a statistically mean-
ingful correlation of variant properties and aggregation Recent studies have revealed that A␤ mediated
propensities. A second set of aggregation propensi- neurotoxicity is likely to be conformation and envi-
ties was derived from Betts et al. [78] where ThT ronment dependent [43, 87]. Thus, to understand at the
intensity was measured on agitated amyloid samples molecular level the conformational and environmental
rather than a conventional static ThT binding assay, dependences of aggregation propensities and cytotox-
reporting the reverse time of maximal ThT binding in icities, four NMR structures (A␤42 : 1IYT [64], 1Z0Q
minutes. Times of half maxima have been compiled [65] and A␤40 : 1BA4 [66], 2LFM [67]) with vari-
as 110 (WT), 130 (A21G), 18 (E22G), 80 (E22K), 60 able environment and conformations were employed
(E22Q), and 50 (D23N) where the reverse of these in this study. We calculated the RMSD values (alpha-
numbers were computed and normalized to WT = 1 in carbon-based) for each conformer in the ensembles
this study (Supplementary Table 13). As shown below, using the first (top score) conformation as reference
both data sets provide statistically significant corre- (Supplementary Tables 1–4). The RMSD values for
lations versus the computed aggregation propensities these four structures ranged between 2.5 Å to 11.8 Å
and the trends are in both cases meaningful, in terms of (Supplementary Tables 1–4), where the most inter-
the changed chemical properties. Thus, despite known esting variations mainly occur in the hydrophobic,
shortcomings of the ThT assay, the comparison and disordered C-terminal part of the molecules. This
analysis done here shows that these experiments can part gives rise to most of the conformational varia-
be meaningfully compared and used together. tion, enabling us to study aggregation propensity as a
M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids 219

function of disorder and exposure mainly due to this Figure 2 shows the correlation between experi-
region. mental and computed aggregation propensities of A␤
variants, divided into structure type. The two panels to
the left show results with a 5 Å radius, and the panels to
Computed versus experimental aggregation the right show the results using 10 Å. Results are shown
propensities using all four different structures 1IYT [64] (A␤42 ;
Fig. 2A, B), 1Z0Q [65] (A␤42 ; Fig. 2C, D), 1BA4 [66]
A number of genetic variations in A␤PP that reside (A␤40 ; Supplementary Fig. 2A, B), and 2LFM [67]
within the A␤ region have been related to FAD or (A␤40 ; Supplementary Fig. 2C, D).
cerebral amyloid angiopathy, but associated with A␤ The computed aggregation propensities obtained
deposits [88, 89]. Data on these variants have been from the different structures correlate substantially, as
compiled in Supplementary Table 13. The changed shown in Supplementary Figure 1, but there are also
chemical properties could affect the processing of distinct variations due to conformational differences
A␤PP and thus, the produced amyloid levels and iso- in the PDB structures. These variations reflect that the
form ratios. However, the mutations will also directly structures differ, as shown in Fig. 1, and were obtained
change the biochemical properties of the amyloids at variable conditions, with 1IYT and 1Z0Q having
as shown in multiple studies of their aggregation 20% and 70% water, respectively, while 1BA4 was
propensity and toxicity, as compiled in Supplemen- measured in a water-micelle mixture and 2LFM was
tary Table 13. These chemical properties, notably their obtained in 100% water. These variations are of inter-
reported aggregation propensities and toxicities, are est because they allow an estimate of the structural
the subject of the present work. features that best describe the experimental data.

Fig. 2. Correlation between computed and experimental aggregation propensities from the first data set [79, 81, 84]: A) using 1IYT and R = 5 Å;
B) using 1IYT and R = 10 Å; C) using 1Z0Q and R = 5 Å; D) using 1Z0Q and R = 10 Å.
220 M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids

We have considered R2 > 0.3 significant for discus- residues both increase absolute aggregation propen-
sion, and a traditional choice of p-value < 0.05 was sities and could also influence the relative aggregation
applied. This implies that the correlation observed propensities of the different A␤ variants.
is less than 5% likely to have occurred by coin- Thus, the SAP method gives encouraging results
cidence, and this confidence basis was achieved in in particular using R = 10 Å. For the specific isoform
several correlations, and thus used as threshold [90]. A␤42 represented by experimental data, R2 = 0.49 and
Figures 2A–D show that the computed aggregation R2 approaches the accuracy of specifically fitted mod-
propensities A␤42 genetic variants are in good agree- els such as the Chiti-Dobson model [91]. The trend
ment with experimental data using both R = 5 Å (1IYT; in aggregation propensities are produced with high
R2 = 0.39, p = 0.04; 1Z0Q R2 = 0.37; p = 0.05) and statistical significance at 99% confidence, showing
R = 10 Å (1IYT; R2 = 0.49, p = 0.02; 1Z0Q; R2 = 0.70; that computed SAP values can provide substantial
p = 0.002). descriptive and interpretative power to the chal-
Somewhat weaker correlations were found for lenge of understanding molecular causes of amyloid
the A␤40 structures 1BA4 and 2LFM both using aggregation.
R = 5 Å (1BA4; R2 = 0.35; p = 0.05; 2LFM; R2 = 0.23; Figure 3 shows the same analysis performed using
p = 0.13) and R = 10 Å (1BA4; R2 = 0.34; p = 0.06; the second experimental data set by Betts et al. [78].
2LFM; R2 = 0.32; p = 0.07) (Supplementary Material, The computed aggregation propensities were in even
Supplementary Fig. 2A–D). This fits well with the fact better agreement with these experimental data and
that the experimental aggregation propensities were significant at the 95% level for all R = 10 Å compu-
measured on the A␤42 isoform, since the last two tations. Trends obtained were strong both with 1IYT

Fig. 3. Correlations between computed and experimental aggregation propensities from the second data set [78]: A) using 1IYT and R = 5 Å;
B) using 1IYT and R = 10 Å; C) using 1Z0Q and R = 5 Å; D) using 1Z0Q and R = 10 Å.
M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids 221

(R2 = 0.80, p = 0.02 using either R = 5 or 10 Å, Fig. 3A, methodology, but they also show that aggregation
B) and 1Z0Q (R2 = 0.58; p = 0.08 using R = 5 Å and propensities are quite conformation-dependent; an
R2 = 0.81; p = 0.02 using R = 10 Å) (Fig. 3C, D). For observation that resonates well with recent experimen-
1BA4 and 2LFM, correlations were likewise signifi- tal findings that cytotoxicity of amyloids is likely to be
cant at the same levels (Supplementary Figure 3). structure-dependent [43, 87]. 2LFM (Supplementary
The strongest correlations were generally obtained Fig. 2C, D) is the least structured conformation among
when using R = 10 Å and these results were obtained the four structures we studied and showed the weakest
for both data sets. Furthermore, the 1Z0Q structure correlation to experimental aggregation propensities,
revealed the highest overall correlation and is likely suggesting that this A␤40 structure differs substantially
to represent the most realistic structure in terms of the from the A␤42 conformations measured experimen-
aggregation experiments; it is a mixed helix-disorder tally.
structure. Thus, the computed aggregation propensities
of the variants using 10 Å and the 1Z0Q structure as Reducing hydrophilic surface is a main
basis gave correlations of R2 = 0.70; p = 0.002 (Fig. 2D) determinant of amyloid aggregation
and R2 = 0.81; p = 0.015 (Fig. 3D), which is as accurate
as linear 3-parameter models with specific parameter- To understand the drivers of amyloid aggregation in
ization [91]. more detail, we further tested whether simple proper-
The computations thus show that one can ties of amyloid variants correlated with experimental
obtain statistical significant prediction of aggregation aggregation propensities. We found that experimen-
propensities for amyloid variants using the applied tal aggregation propensities were not correlated

Fig. 4. Correlation between experimental aggregation propensities from the first data set [79, 81, 84] and computed hydrophilic surfaces of
amyloid variants: A) using structure 1IYT; B) using structure 1Z0Q; C) using structure 1BA4; D) using structure 2LFM.
222 M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids

Fig. 5. Correlation between experimental aggregation propensities from the second data set [78] and computed hydrophilic surfaces: A) using
1IYT; B) using 1Z0Q; C) using 1BA4; D) using 2LFM.

significantly with computed hydrophobic surface and (R2 = 0.32; p = 0.07, Fig. 4C), and 2LFM (R2 = 0.32;
total solvent surface for the first data set (Supplemen- p = 0.07, Fig. 4D). Significant correlation was found
tary Material, Supplementary Figs. 4 and 5), although when using the 1Z0Q structure (R2 = 0.40; p = 0.05,
the trends show that they have some importance. The Fig. 4B).
same conclusions were obtained using the data by Figure 5 shows a similar regression analysis using
Betts et al. [78] (Supplementary Figs. 6 and 7), i.e., the experimental aggregation propensities reported by
hydrophobic surface contributes but is a partial descrip- Betts et al. [78]. In this case, computed hydrophilic
tor for all 8 regressions shown in Supplementary Figs. 4 surface produced highly significant correlations: 1IYT
and 6. gave R2 = 0.87; p = 0.007 (Fig. 5A), 1Z0Q gave
Figures 4 and 5 show the linear regression plots R2 = 0.64; p = 0.06 (Fig. 5B), 1BA4 had R2 = 0.82;
for aggregation propensities and hydrophilic surfaces p = 0.01 (Fig. 5C), and 2LFM had R2 = 0.83; p = 0.01
of all the A␤ species. Figure 4 shows the correla- (Fig. 5D). For data in Fig. 4, the A␤ variants D678N
tion between experimental aggregation propensities (D7N; a normalized propensity versus WT of 3.4) and
reported by Murakami et al. [79], Zhou et al. [81], A692G (A21G; 0.07) are outliers in the scatter plots.
and Hori et al. [84], and computed hydrophilic sur- For the second data set in Fig. 5, outliers are less sub-
faces of the A␤ species: This property showed some, stantial.
but not significant at the 95% confidence level, corre- From these correlation coefficients and p-values
lation with reported aggregation propensities for the (see Supplementary Tables 18–21 for numerical val-
structures 1IYT (R2 = 0.34; p = 0.06, Fig. 4A), 1BA4 ues of these parameters), we conclude that reduced
M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids 223

hydrophilic surface is a determinant in describing the cally consistent with aggregation contributing to cell
aggregation propensity of the known disease-related toxicity, the first such link supported by statistical sig-
A␤ variants. The negative correlations are physically nificance from regression analysis of available data.
meaningful as they imply that peptide aggregation is Even with heterogeneity in lab protocols and with the
determined largely by reduced hydrophilic surface, variations in chemical structure of the amyloids that
and reduced hydrophilic surface as seen in some vari- necessarily create noise in the data sets, these corre-
ants strongly correlates with increased aggregation lations persist in the majority of cases studied and are
propensity. thus very unlikely (as also seen from the p-values) to
have occurred coincidentally.

Experimental toxicities correlate with

structure-dependent aggregation propensities Chemical causes for the high aggregation
propensity of Aβ variants
The reported cytotoxicities of A␤ variants [79, 80],
as quantified by their EC50 values, are thought to relate Increase in hydrophobic exposure is considered
to aggregation of amyloids [44], although no statisti- one of the major driving forces causing A␤ variants
cally significant correlation from analysis of general to aggregate [92–94]. For all the A␤ structures, the
available data has yet been described. To investigate computed hydrophobic surface area provides some
whether we could identify a simple cause of cytotox- correlation to aggregation propensity but is not statisti-
icities of genetic A␤ variants, we correlated known cally significant by itself (Supplementary Figures 4 and
EC50 values for these variants against computed prop- 6), consistent with general view that the hydrophobic-
erties, i.e., hydrophobic surface, hydrophilic surface, ity only constitutes part of the chemical driving force
total surface, and SAP. None of these other proper- toward aggregation [91, 94].
ties showed any significant correlation by themselves Previous experimental investigations of the aggre-
(Supplementary Figs. 8–10). gation propensities of A␤ variants (Supplementary
Figure 6 shows the correlation plots of computed Table 13) have suggested that E22G and D7N variants
aggregation propensities vs. normalized experimen- are most aggregation prone compared to the WT,
tal EC50 values, with the left panels again showing whereas the A21G variant was found to have lower
R = 5 Å and the four right panels showing R = 10 Å. aggregation tendency compared to the WT, i.e.,
The correlations were again dependent on structure E22G > D7N > D23N > E22Q ≥ H6R > A2V = D7H >
used: With 5 Å radius, 1IYT gave R2 = 0.36, p = 0.05 E11K > E22K > WT > A21G (Supplementary Table 13).
(Fig. 6A); 1Z0Q gave R2 = 0.55; p = 0.009 (Fig. 6C); The latter data point for A21G is an outlier in the
1BA4 gave R2 = 0.60; p = 0.02 (Fig. 6E), and 2LFM regression and has an unusually low aggregation
gave R2 = 0.20; p = 0.23 (Fig. 6G).With R = 10 Å, propensity versus WT. While this variant has reduced
1IYT gave R2 = 0.52, p = 0.01 (Fig. 6B); 1Z0Q gave hydrophobic surface by mutation to the small,
R2 = 0.31; p = 0.08 (Fig. 6D); 1BA4 gave R2 = 0.50; hydrophilic glycine, the error in the regression
p = 0.04 (Fig. 6F); and 2LFM gave R2 = 0.44; p = 0.05 could be due to introduction of glycine, which can
(Fig. 6H). From all figures, it can be seen that smaller produce problems in computed structures due to real
experimental EC50 value, corresponding to higher tox- co-localization of water not present in the WT-based
icity, correlates positively with computed aggregation structure [95].
propensity. This correlation is robust against varia- The E22G variant exhibits the highest aggregation
tions in structure, providing theoretical insight into tendency both experimentally and in our computations,
the relationship between aggregation and cell toxic- and is the second most toxic variant so far characterized,
ity of amyloids. With this correlation at hand, one as measured from EC50 values [79] (Supplementary
can identify chemical properties important not only Table 13). A link to the reduction of hydrophilic surface
for aggregation propensity but also for toxicity. (Figs. 7 and 8) can be seen numerically from Supple-
From this analysis, we conclude that experimentally mentary Tables 9 to 12, irrespective of structures and
reported toxicities of the various amyloid variants cor- methods used in the computations. The hydrophilic sur-
relate significantly to structure-dependent aggregation face area is reduced from 1257 Å2 in the WT (Fig. 7A) to
propensities of the species. Five of the eight correla- 1174 Å2 in the E22G variant (Fig. 7B) when being in the
tions in Fig. 6 are significant at the 95% confidence 1IYT conformation (Supplementary Table 9). When in
level, and the direction of the correlation is physi- a 1Z0Q-like conformation, this change is from 1189 Å2
224 M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids

Fig. 6. Correlation between experimental EC50 [79, 80] and computed aggregation propensities with probe radii (R) of 5 Å and 10 Å: A) using
1IYT and R = 5 Å; B) using 1IYT and R = 10 Å; C) using 1Z0Q and R = 5 Å; D) using 1Z0Q and R = 10 Å; E) using 1BA4 and R = 5 Å; F) using
1BA4 and R = 10 Å; G) using 2LFM and R = 5 Å; H) using 2LFM and R = 10 Å.
M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids 225

Fig. 7. Hydrophilic surface area of WT and mutants of A␤42 : A) and B) using 1IYT structures; C) and D) using 1Z0Q. The mutated residue is
shown in sticks while the affected molecular surface is represented in green color. Secondary structure is represented with blue N-terminus and
red C-terminus. The picture was made using D.S. 4.0 visualizer.

Fig. 8. Hydrophilic surface of WT and E22G mutants of A␤40 : A) and B) using 1BA4C) and D) using 2LFM. The mutated residue is shown
in sticks while the affected surface is represented in green. Secondary structure is represented with blue N-terminus and red C-terminus. The
picture was made using D.S. 4.0 visualizer.
226 M.K. Tiwari and K.P. Kepp / Modeling Aggregation and Toxicity of Amyloids

to 1133 Å2 (Fig. 7C, D; Supplementary Table 10); with ACKNOWLEDGMENTS

1BA4 the change is from 1163 Å2 to 1091 Å2 (Fig. 8A,
B; Supplementary Table 11), and with 2LFM the The authors acknowledge the Technical University
reduction is from 1429 Å2 to 1350 Å2 (Fig. 8C, D; Sup- of Denmark for providing a Hans Christian Ørsted
plementary Table 12). E22G gave the highest computed (HCØ) fellowship to MKT.
aggregation propensity using both R = 5 Å and 10 Å Authors’ disclosures available online (http://j-alz.
across all the structures. com/manuscript-disclosures/15-0046r2).
While the hydrophilic surface area seems relevant to
amyloid aggregation, the molecular mode of toxicity of
amyloids remains under debate [96, 97], but it has been SUPPLEMENTARY MATERIAL
suggested that membrane interactions are involved
[36]; such a molecular mode of toxicity is consistent The supplementary material is available in the
with an increase in structure-dependent aggregation electronic version of this article:
propensity of specific conformations of the amyloids, 10.3233/JAD-150046.
either directly in the monomer forms or in oligomers
that have been formed from monomers with high
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