Cholesterol Changes the Mechanisms of AÎ² Peptide Binding to the

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Cholesterol changes the mechanism of A# peptide binding to the DMPC bilayer Christopher Lockhart, and Dmitri K. Klimov J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00431 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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Journal of Chemical Information and Modeling

CHOLESTEROL CHANGES THE MECHANISMS OF Aβ PEPTIDE BINDING TO THE DMPC BILAYER Christopher Lockhart and Dmitri K. Klimov

∗

School of Systems Biology, George Mason University, Manassas, VA 20110

E-mail: [email protected]

Abstract Using isobaric-isothermal all-atom replica-exchange molecular dynamics (REMD) simulations, we investigated the equilibrium binding of Aβ 10-40 monomers to the zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer containing cholesterol. Our previous REMD simulations, which studied binding of the same peptide to the cholesterolfree DMPC bilayer, served as a control, against which we measured the impact of cholesterol. Our ndings are as follows. First, addition of cholesterol to the DMPC bilayer partially expels the Aβ peptide from the hydrophobic core and promotes its binding to bilayer polar headgroups. Using thermodynamic and energetics analyses, we argued that Aβ partial expulsion is not related to cholesterol-induced changes in lateral pressure within the bilayer, but is caused by binding energetics, which favors Aβ binding to the surface of the densely packed cholesterol-rich bilayer. Second, cholesterol has a protective eect on the DMPC bilayer structure against perturbations caused by Aβ binding. More specically, cholesterol reduces bilayer thinning and overall depletion of bilayer density beneath the Aβ binding footprint. Third, we found that the Aβ peptide contains a single cholesterol binding site, which involves hydrophobic C-terminal 1

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amino acids (Ile31-Val36), Phe19 and Phe20 from the central hydrophobic cluster, and cationic Lys28 from the turn region. This binding site accounts for about 76% of all Aβ -cholesterol interactions. Because cholesterol binding site in the Aβ 10-40 peptide does not contain the GXXXG motif featured in cholesterol interactions with the transmembrane domain C99 of the β -amyloid precursor protein, we argued that the binding mechanisms for Aβ and C99 are distinct reecting their dierent conformations and positions in the lipid bilayer. Fourth, cholesterol sharply reduces the helical propensity in the bound Aβ peptide. As a result, cholesterol largely eliminates the emergence of helical structure observed upon Aβ transition from a water environment to the cholesterolfree DMPC bilayer. We explain this eect by the formation of hydrogen bonds between cholesterol and the Aβ backbone, which prevent helix formation. Taken together, we expect that our simulations will advance understanding of a molecular-level mechanism behind the role of cholesterol in Alzheimer's disease.

Introduction The exact role of A β peptides in Alzheimer's pathogenesis has not been rmly established. Several studies have linked A β peptides to neuronal loss, chronic inammation, and immune system response to bacterial or fungal infections.

15

The most abundant alloform of A β is the

40-mer species, A β 1-40, produced as a result of cleavage of the by

β - and γ -secretases.

β -amyloid

precursor protein

A β peptides are highly amyloidogenic and their aggregation proceeds

through a complex sequence of structural transitions, starting with the oligomerization of monomers.

6

Regardless of the specic role played by A β peptides as a pathological agent

or antimicrobial peptide, it is well known that A β aggregates, even as small as dimers, are cytotoxic species capable of inducing neuronal death.

7

Interactions of A β peptides with lipid bilayers have been extensively investigated via experiments

816

and simulations.

17,18

Depending on their concentration, A β peptides may

exist on the membrane surface either in monomeric

13

or low-order oligomeric

2


12

forms. In

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general, small oligomers display a higher binding anity compared to other species, including monomers, large oligomers, and brils.

19,20

Electron density analysis of X-ray diraction data

has shown that A β peptides can penetrate deep into lipid hydrophobic cores of the bilayers potentially aecting their structural integrity.

9,21

Indeed, experimental data suggest that the

bilayer structure is disrupted to a larger extent by the penetration of A β peptides into the bilayer core rather than by their binding to the bilayer surface.

8,15

It has been demonstrated

that Aβ peptides can even completely dissociate zwitterionic bilayers converting them into micelles, of ions.

22

although their impact is typically less severe resulting in an increased permeability

14,23

Previous studies have suggested dierent mechanisms of ion permeation through

lipid bilayers. For example, A β aggregates may promote the permeability of ions (such as Ca

2+ 24 23,25 ) by forming structured pores. In fact, theoretical considerations and AFM ex-

periments have shown that A β peptides may indeed form annular ordered oligomers with a central ion channel embedded in lipid bilayers.

12,26

An alternative permeation mechanism

is associated with mobile, loosely organized clusters of A β monomers, which create sucient structural inhomogeneities in bilayers to enhance ion permeation.

22,27,28

a similar scenario may explain ion trac promoted by antimicrobial peptides.

Interestingly,

29

One of the ubiquitous factors that may aect A β cytotoxicity is cholesterol. Although it is a major component of neuronal cell membranes, the role of cholesterol in A β cytotoxicity is highly controversial. There is strong evidence supporting the protective role of cholesterol as its depletion in brain tissues may enhance A β cytotoxic eects leading to neurodegeneration.

30

One explanation for this observation is that cholesterol orders lipid bilayers hindering

the insertion of A β peptides and reducing ion permeability.

31

At the same time, there are

equally compelling indicators that, because cholesterol orders lipid bilayers, it turns them into a template for binding A β peptides that only facilitates their aggregation and cytotoxicity.

32,33

Several previous investigations have analyzed cholesterol binding to A β variants,

such as the short fragment A β 22-35, or Aβ precursors, including the C-terminal fragment C99 of the

β -amyloid

precursor protein.

3436

They identied a combination of hydrophobic

3



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amino acids from the C-terminal and central hydrophobic cluster together with a few anionic amino acids as putative cholesterol binding locations. However, the described binding sites may dier from those characterizing cholesterol binding to A β 1-40 peptides. This assertion is supported by the fact that the A β peptide represents only a half of the C99 transmembrane section and is likely to adopt the conformations dierent from C99 within the bilayer. Therefore, the mechanisms of cholesterol binding to A β peptides and C99 may be distinct and the eect of cholesterol on A β may be entirely dierent from that observed for C99. To gain new insight into the role of cholesterol in A β -bilayer interactions, we used allatom explicit membrane and water models coupled with replica-exchange molecular dynamics (REMD) to probe the equilibrium binding of A β 10-40 peptides to the zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer containing cholesterol. As a control, we employed our previous REMD simulations, which examined the binding of the same A β 10-40 peptides to the cholesterol-free DMPC bilayer.

3739

In those studies, we showed that binding to the

DMPC bilayer changes the A β secondary structure by promoting the formation of stable helix in the C-terminal, which is not present in water.

40

More importantly, we observed

that the Aβ central hydrophobic cluster and, particularly, the C-terminal penetrate deep into the core of the cholesterol-free DMPC bilayer. We also found that A β peptide binding causes considerable thinning and structural perturbation of the bilayer. To our knowledge, those simulations were the rst to demonstrate the feasibility of exhaustive REMD sampling of Aβ peptide binding to lipid bilayers in all-atom resolution and in explicit water, which provides the results consistent with NMR data.

41

Therefore, our previous studies oer an

excellent control to investigate the impact of cholesterol on the mechanisms of A β binding and penetration into the zwitterionic lipid bilayer. This investigation constitutes the main objective of the current work.

4


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Methods Simulation protocol:

Isobaric-isothermal all-atom explicit-solvent replica-exchange molec-

ular dynamics (REMD) simulations were used to probe the binding of A β 10-40 peptides to the zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid bilayer with cholesterol. Our previous simulations investigating binding of A β 10-40 to the cholesterol-free DMPC bilayer were used as a control.

37,38

Selection of the same amino-truncated A β 10-40 peptides in both

simulations allows us to directly examine the impact of cholesterol on the interactions of this peptide with the lipid bilayer. It is also worth mentioning that A β 10-40 peptides are naturally occurring cytotoxic and amyloidogenic species lipid bilayers

37,38

4245

and their interactions with the

bear similarities with those reported experimentally for the full-length

Aβ 1-40 peptide binding to SDS micelles or DMPC bilayers.

10,41

The selection of simple,

one-component (DMPC) lipid bilayer makes it easier to delineate the role of cholesterol in Aβ binding to the bilayers. In the simulation system, two A β 10-40 monomers were placed on either side of the lipid bilayer, which contained 36 DMPC lipids and 28 cholesterol molecules per leaet (Fig. 1). Therefore, the molar fraction of cholesterol was

x ≈ 0.44,

which approximately matches

experimentally-determined in vivo molar ratios of cholesterol to lipids in the presence of Aβ .

32

It is useful to note that placing two peptides on the opposite sides of the bilayer

allows us to avoid artifacts associated with dierent lateral pressures in the leaets, which may develop if only one A β 10-40 peptide binds to the bilayer. solvated with 4372 water molecules. the net charge on A β peptides.

46

The entire system was

Two sodium counterions were added to neutralize

Aβ featured acetylated and amidated termini as well as

uncharged histidines corresponding to neutral pH. The initial dimensions of the simulation box were 59 Å x 59 Å x 94 Å. The simulations utilized the program NAMD, CMAP corrections,

48

47

the CHARMM22 protein force eld with

and the CHARMM36 lipid force eld.

49

REMD used 40 temperatures

distributed exponentially from 320 to 430 K. Exchanges were attempted every 2 ps.

5


The


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(a) Aβ10-40: Y10EVHHQKLVFF20AEDVGSNKGA30IIGLMVGGVV40 R1 (b)

R2

R3

R4

GL3

GL5

GL1 GL4

GL2 GC2

GC3 (c)

GC1

(d)

zc 0 -zc

Figure 1: (a) A β 10-40 sequence with the four sequence regions R1-R4 distinguished by color. (b) A DMPC lipid is divided into ve structural groups: choline (GL1), phosphate (GL2), glycerol (GL3), and two fatty acid tails (GL4 and GL5). The polar lipid headgroups include GL1-GL3. The bilayer hydrophobic core is composed of GL4 and GL5. Phosphorus atom is shown as a large purple sphere. (c) Cholesterol is divided into the hydroxyl group (GC1), steroid rings (GC2), and the hydrocarbon tail (GC3). Atom C13 is shown as a large grey sphere. (d) A snapshot of cholesterol-rich DMPC bilayer with bound A β peptides. DMPC and cholesterol molecules are in tan and green, respectively.

The centers of mass of lipid

phosphorus P (purple spheres) and cholesterol C13 (grey spheres) atoms in each leaet are approximately restrained at

±zc (zc = 18.26Å). Aβ

peptides (in orange) bind to the opposite

leaets of the bilayer. Water is shown in blue. For clarity, we omitted ions.

integration step was set to 1 fs. In each replica, temperature was maintained using underdamped Langevin simulations of virtual solvent with the damping coecient

γ

= 5 ps

−1

.

Pressure was held constant using the Langevin piston method, and a semi-isotropic coupling

6


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scheme was employed, which adjusts the separately from the

z

x

and

y

box dimensions (along the bilayer plane)

dimension. Simulations were performed with periodic boundary con-

ditions. Electrostatic interactions were computed using Ewald summation, whereas van der Waals interactions were smoothly switched o in the interval from 8 to 12 Å. All hydrogen covalent bonds were constrained by the ShakeH algorithm. Following our previous studies,

37,50,51

two sets of restraints were added to the system to

prevent disintegration of the bilayer at high REMD temperatures and to block A β aggregation. The rst set were harmonic restraints with the force constant

k

= 17.17 kcal/mol/Å

2

that approximately xed the center of mass of lipid phosphorus atoms and cholesterol C13 atoms in a leaet at the distance force constant

zc

from the bilayer midplane (Fig. 1c). To determine the

k and the distance zc we performed separate A β - and restraint-free simulations

of the DMPC bilayer with cholesterol at 330 K, from which we computed the z-position uctuations of the center of mass of lipid phosphorus atoms and cholesterol C13 atoms and its average position

zc .

The value of

k was then chosen to reproduce those observed uctuations.

In our previous studies we showed that such restraints produce minimal perturbations in the bilayer structure. 4 Å of the

z

52

The second set of restraints acted on A β atoms when they came within

periodic boundary. Technically, they were implemented as harmonic restraints

with the force constant

k

= 10 kcal/mol/Å

2

that act only on A β atom

330 K, these combined restraints contributed, on average,

1.5kcal/mol

z

coordinates. At

to the total potential

energy and therefore are expected to have a minimal impact on thermodynamic quantities of interest. During our REMD simulations, the probability for two A β peptides to interact either across the

z

boundaries or across the bilayer was negligible (0.0% at 330 K). As a

result, Aβ peptides bind independently to the bilayer. In total, ve REMD trajectories were produced. Individual trajectories were started from arbitrary initial conformations of A β peptides. A thorough analysis of REMD convergence is presented in the Supporting Information. As a measure of replica exchange performance, we computed the average replica exchange acceptance rate across all temperatures as 23%.

7



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Each replica in a trajectory was simulated for 20 ns. Therefore, collectively, we generated 4

µs

of sampling across all temperatures and trajectories. Approximately 1

µs

of sampling at

the beginning of REMD trajectories was discarded as unequilibrated resulting in of equilibrium simulation time.

However, because the two peptides bind to the bilayer

independently, the eective sampling per peptide is doubled to 6

Computation of structural probes: our previous studies.

37,38,50,51

τsim = 3µs

µs.

The analysis of simulation data largely followed

Specically, we divided the A β sequence into four regions: the

hydrophilic N-terminal (R1, residues 10-16), the central hydrophobic cluster (R2, residues 1721), the hydrophilic turn region (R3, residues 22-28), and the hydrophobic C-terminal (R4, residues 29-40) (Fig. STRIDE.

53

1a).

Peptide secondary structure was computed using the program

An amino acid was in a helical state if STRIDE assigns an

α-, 310 -,

or

π -helix.

Intrapeptide contacts were assessed by reducing the peptide to side chain geometrical centers. Then, two side chains were assumed in contact if the separation between their geometrical centers was less than 6.5 Å, which approximately corresponds to the exclusion of water between them. Likewise, we computed the interactions between amino acids and lipids or cholesterol. To this end, DMPC lipids and cholesterol were divided into several structural groups as indicated in Fig. 1b,c. Contacts occurred between these groups and amino acid side chains if the distance between their geometrical centers was less than 6.5 Å. Hydrogen bonds between A β and the bilayer were assigned by applying standard geometric criteria.

54

An Aβ amino acid is bound to the bilayer if it makes a contact with at least one DMPC or cholesterol structural group. We assessed the structure of the bilayer by computing the number density of bilayer heavy atoms

nBL (r, z) as a function of the distance r

plane) and the distance

z to the bilayer midplane.

along the bilayer normal

nc (z), respectively). distributions

P (z; i)

z

to the Aβ center of mass (in the bilayer

We also considered number density proles

for peptide, lipid, and cholesterol heavy atoms ( np (z),

nl (z),

and

To characterize A β binding to the bilayer, we computed the probability of the occurrence of amino acids

i

at the distance

8


z

from the bilayer

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midplane. From

P (z; i), we derived the probabilities Pi (i) for amino acids i to insert into the

hydrophobic core, i.e., to occur below the center of mass of DMPC phosphorus atoms in a leaet

zP .

Note that

zP

is treated as an approximate boundary between bilayer hydrophobic

core and polar headgroups. Similarly,

Ps (i) was dened as the probability for amino acid i to

bind to the bilayer surface, i.e., to localize within the headgroup region ( zP , zP +6.5 Å). Lipid ordering was quantied by computing the lipid carbon-deuterium order parameter

sn − 2

SCD

for

lipid fatty acid tails (GL4 in Fig. 1b).

To identify cholesterol binding sites in the A β peptide, we applied the method developed previously by our group.

55

Briey, we dene a binding site as a set of A β amino acids

satisfy two conditions. First, amino acids the number of contacts with cholesterol

i

< Cc,cb (m, n) >

that

must form strong contacts with cholesterol, i.e.,

< Cc (i) >≥0.4maxm (< Cc (m) >),

sents an Aβ amino acid. Second, amino acids cholesterol-mediated cross-bridging, i.e.,

i

i and j

where

m

repre-

from a binding site must participate in

< Cc,cb (i, j) >≥0.2maxm,n (< Cc,cb (m, n) >),

where

is the number of cholesterol molecules interacting simultaneously with (i.e.,

cross-bridging) amino acids

m

and

n.

Thermodynamic averages of structural quantities (denoted by ) were computed using the multiple histogram method

56

modied for isobaric-isothermal simulations.

57,58

Fur-

thermore, these computed quantities reect the averages over two A β peptides binding to the opposite sides of the bilayer. For consistency with our previous work,

37,38,50,51

simulation

results are reported at 330 K.

Results Aβ conformational ensemble To evaluate the eect of cholesterol on the A β conformational ensemble, we rst computed the peptide secondary structure. When the A β peptide binds to the cholesterol-rich DMPC bilayer, the overall fractions of helix

< H >,

turn

< T >,

and random coil

9


< RC >

were


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0.20±0.04, 0.53±0.02, and 0.26 ±0.02, respectively. and excluded from further analysis.

Page 10 of 39

β

The fraction of

states is negligible

For comparison, the corresponding secondary struc-

ture fractions for A β bound to the pure DMPC bilayer were 0.39 ±0.03, 0.30±0.03, and 0.31±0.01.

37

These results demonstrate that cholesterol reduces the helix propensity almost

two-fold, while enhancing the turn fraction. To get additional insight, we plot in Fig. 2 the probabilities of helix

< H(i) > and turn < T (i) > conformations for each amino acid i.

Fig.

2a demonstrates a signicant reduction in helix for more than half of amino acids in the peptide bound to the cholesterol-rich DMPC. Indeed, in cholesterol-free DMPC simulations, 11 amino acids adopted stable helix structure (
≥0.5), which occurred in R3 (23-26)

and R4 (31-37) regions. In contrast, when A β binds to the cholesterol-rich DMPC bilayer, no residue forms stable helix. More subtle changes in the secondary structure are reported in Table S1 in the Supporting Information.

This table shows that, in the cholesterol-rich

DMPC system, helix is actually enhanced by more than a factor of 2 in the sequence region R1, remains approximately unchanged in R2, and is signicantly reduced in R3 and R4 (by approximately three-fold). The mechanism of helix destabilization is rationalized in the Discussion. Fig. 2b further shows that, whereas only ve amino acids (12-14 in R1, 21 in R2, and 22 in R3) formed stable turn (
≥ 0.5)

for the pure DMPC bilayer, 14 amino acids

(20-27 in R2-R3, and 31-36 in R4) adopt stable turn in A β bound to the DMPC bilayer with cholesterol. These results are corroborated by Table S1 showing that cholesterol enhances turn formation in R2-R4 by factors of 1.2, 1.7, and 5.3, respectively.

Finally, for the A β

peptide bound to the cholesterol-rich DMPC bilayer, random coil structure is reduced (Table S1), particularly in the R1 and R2 regions. In summary, we found that cholesterol alters the secondary structure of A β bound to the bilayer by destabilizing helix and promoting turn conformations. Changes in secondary structure induced by cholesterol correlate with tertiary reorganization. Although the total numbers of intrapeptide contacts

10


in Aβ peptides bound

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(a)

(b)

Figure 2: Secondary structure propensities for A β amino acids

H(i) > and (b) turn propensity < T (i) >.

i:

(a) helix propensity

,

− j| ≥5),

which occur between amino acids

i

and

j

increases from 11.1 ±0.6 to 14.1±0.9, respectively.

Accordingly, for the DMPC bilayer with cholesterol, approximately 50% of all A β intrapeptide contacts are long-range, whereas, for the pure DMPC bilayer, this fraction is reduced to 39%.

To examine the detailed changes in tertiary interactions, we plot in Fig.

dierence contact map

< ∆C(i, j) >

3 the

comparing A β peptides bound to the DMPC bilayers

11



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with and without cholesterol. tacts (|

Page 12 of 39

Table S2 presents the 15 most aected intrapeptide con-

< ∆C(i, j) > | ≥0.35).

It is seen that most of the destabilized contacts (7 out of

10) are short-range, reecting helix disruption. Cholesterol also destabilizes two long-range hydrophobic contacts between either Phe19 (in R2) or Val24 (in R3) and Ile31 (in R4). However, the most impacted interaction is the salt-bridge Asp23-Lys28, which is formed in Aβ bound to the cholesterol-free DMPC bilayer (
=0.79)

but becomes largely

disrupted with the addition of cholesterol (0.17). Interestingly, Table S2 does not reect well an increase in the fraction of long-range interactions reported above as only two out of ve stabilized contacts are long-ranged. Therefore, the enhancement of long-range contacts must result from the net stabilization of multiple weak interactions. Indeed, the analysis of the dierence contact map in Fig. 3 reveals that 60% of all possible long-range contacts become more stable due to cholesterol.

Consequently, cholesterol overall promotes long-range A β

interactions as implied by the computation of the number of long-range contacts

< CLR >

above. This restructuring of A β tertiary interactions does not aect the compactness of the peptide, i.e., for both bilayers, the radius of gyration

< Rg >

is 14.8 Å.

Aβ -lipid interactions Our REMD simulations allow us to probe the equilibrium distribution of A β amino acids along the bilayer normal. The corresponding results are presented in Fig. 4, which visualizes the probabilities

P (z; i) for an amino acid i to be at the distance z

from the bilayer midplane

(z =0). The summary is also given in Table S3, which reports the probabilities,

Ps (k),

for amino acids from the sequence regions

k =R1-R4

Pi (k)

and

to be inserted or surface-bound

(see Methods). Collectively, Fig. 4 and Table S3 elucidate several trends. First, out of all Aβ regions only the hydrophobic R4 is classied as inserted ( Pi (R4)

≥ 0.5).

For comparison,

both hydrophobic regions, R2 and R4, are inserted into the cholesterol-free DMPC bilayer. Most importantly, all A β sequence regions

k =R1-R4

cholesterol feature smaller insertion probabilities

Pi (k)

bound to the DMPC bilayer with than for the cholesterol-free DMPC

12


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Figure 3: The dierence contact map

< ∆C(i, j) >

displays changes in A β intrapeptide

i and j caused by cholesterol. We dene < ∆C(i, j) >=< C(i, j) > − < C(i, j) >0 , where the contact maps < C(i, j) > and < C(i, j) >0 are computed for the cholesterol-rich and -free bilayers. The values of < ∆C(i, j) > are color-coded in the inset scale. To ease interpretation, short-range contacts ( |i − j| < 5) are placed above the diagonal, and long-range contacts ( |i−j| ≥ 5) are placed below the diagonal. Regions R1-R4 contacts between amino acids

are colored following Fig. 1a. The gure demonstrates the overall destabilization of shortrange contacts and net enhancement of long-range interactions in the A β peptide bound to the DMPC bilayer with cholesterol.

bilayer. Second, cholesterol reduces the probability of surface binding

Ps (k)

for R1, has a

negligible eect on the R3 region, and promotes surface binding for R2 and R4. Third, due to cholesterol, the probabilities of unbinding

Pu (k)=1 − Ps (k) − Pi (k) are increased for three

out of four Aβ regions (R1-R3, Table S3). Consistent with this analysis, most amino acids in the bilayer with cholesterol (24 out of 31) have their positions

< z(i) >

in Fig. 4 shifted

upward compared to the cholesterol-free DMPC bilayer. The ndings described above are further illustrated in Fig. S6, which presents the number density

np (z)

of Aβ heavy atoms along the bilayer normal

z.

Applying the same denitions

as for inserted and surface bound amino acids, we found that in the DMPC bilayer with cholesterol, 26% of A β atoms are inserted, whereas 37% are surface-bound. The corresponding numbers for the cholesterol-free DMPC bilayer show a reversed preference, with 40% of atoms inserted and 31% surface-bound. Along with the analysis of the probabilities

13


P (z; i),


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Figure 4: The probability distributions distance

z

P (z; i)

for Aβ amino acids

Page 14 of 39

i

to be localized at the

from the bilayer midplane. The probabilities are colored according to the scale

on the right. The average

< z(i) >

positions of amino acids

i

in the DMPC bilayer with

cholesterol are shown by the black line, whereas the red line corresponds to

< z(i) >

for the

cholesterol-free bilayer. The dashed line marks the boundary between bilayer hydrophobic core and polar headgroups. Errors are indicated by vertical bars. Regions R1-R4 are colored following Fig.

1a.

The cholesterol-free distribution

< z(i) >

is shifted 3.88 Å upward to

match the boundaries between hydrophobic core and polar headgroups in both bilayers. The gure shows that the A β bound to the DMPC bilayer with cholesterol is partially expelled from the hydrophobic core compared to the cholesterol-free DMPC bilayer.

these results demonstrate that cholesterol reduces the penetration of A β into the bilayer core, while favoring stronger interactions with the bilayer surface. Additionally, cholesterol increases the fraction of unbound atoms from 29 to 37%. Next, we explore directly the interactions between A β amino acids and lipids or cholesterol. To this end, Fig. 5a presents the A β -lipid contact map

< Cl (i, k) >,

the average numbers of contacts forming between lipid structural groups

i.

k

which reports

and amino acids

This gure and its summary in Table S4 indicate that all A β sequence regions form in-

teractions with the lipid headgroups GL1-GL3. In contrast, only the hydrophobic region R4 forms signicant interactions with the fatty acid tails GL4/GL5. Consequently, for all A β regions, the numbers of interactions with the headgroups far exceed those with the fatty acid tails. Among all residues, cationic amino acid Lys28 forms the strongest interactions with lipid groups, binding to phosphate GL2 and glycerol GL3. Availability of Lys28 for inter-

14


Page 15 of 39

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action with the cholesterol-rich DMPC bilayer is related to the disruption of Asp23-Lys28 salt-bridge reported above. It is important to assess changes in A β -DMPC interactions induced by cholesterol. To this end, Table S4 reveals that, when cholesterol is present, all A β sequence regions form fewer contacts with fatty acid tails (GL4-GL5). Furthermore, three A β regions R2-R4 strengthened interactions with the lipid headgroups (GL1-GL3) of the cholesterol-rich bilayer and only for R1 does the number of contacts with the headgroups remain unchanged.

Overall, in the

cholesterol-rich bilayer, A β peptide forms four-fold more contacts with the lipid headgroups (21.1

± 1.8)

than with the fatty acids ( 5.0

± 1.6).

In the cholesterol-free bilayer, the ratio of

the number of contacts with the headgroups to those with the fatty acids is only two-fold (16.1

± 2.8

vs

8.1 ± 2.3).

(Note that cholesterol-free data are normalized due to the larger

number of lipids in the bilayer.) To get further insight, Fig. 5b displays the dierence contact map

< ∆Cl (i, k) >,

illustrates changes in the interactions between individual A β amino acids tural groups

k

due to cholesterol.

Aβ -DMPC contacts ( |

i

which

and lipid struc-

Using this gure, we identied the ve most aected

< ∆Cl (i, k) > | ≥0.35) listed in Table S5.

contacts (Lys28-GL2 and Lys28-GL3,

< ∆Cl (i, k) >>0)

Two out of three stabilized

reect strong interactions between

cationic Lys28 and the cholesterol-rich bilayer described above. It is also worth noting that, in Table S5, all stabilized interactions are formed with the lipid headgroups and the remaining two, destabilized contacts (Phe20-GL4 and Phe20-GL5) are formed with the bilayer hydrophobic core. Therefore, taken together, our ndings suggest that cholesterol promotes partial expulsion of the A β peptide from the hydrophobic core to the polar bilayer surface. Our analysis also reveals that cholesterol enhances electrostatic interactions between cationic amino acids and DMPC headgroups. Finally, we probe the binding of A β to cholesterol. Using the number of contacts formed by amino acid

i with cholesterol molecules, < Cc (i) >, shown in Fig.

S7 and also considering

Table S4, we assess that A β -cholesterol interactions are mostly restricted to the C-terminal

15



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Page 16 of 39

(a)

(b)

< Cl (i, k) > visualizes the interactions forming between lipid < Cl (i, k) > is color-coded according to the scale on the right. (b) The dierence contact map < ∆Cl (i, k) >=< Cl (i, k) > − < Cl (i, k) >0 compares Aβ -DMPC contact maps obtained with ( < Cl (i, k) >) and without (< Cl (i, k) >0 ) cholesterol. To facilitate the comparison, < Cl (i, k) >0 is normalized to

Figure 5: (a) The contact map structural groups

k

and Aβ amino acids i. The value of

reect dierent numbers of lipids in the bilayers with and without cholesterol. The value of

< ∆Cl (i, k) >

is color-coded according to the scale on the right. Regions R1-R4 are colored

following Fig. 1a. Both panels indicate that cholesterol enhances contacts between A β and lipid headgroups.

residues 31-35, Phe19, Phe20, and Lys28. In descending order, the ve most frequent cholesterol contacts are formed by hydrophobic amino acids Leu34, Gly33, Met35, Ile32, and Ile31. Similar to the analysis of A β -DMPC interactions, we present in Fig. 6a the contact map

< Cc (i, k) >, k.

which visualizes the interactions between amino acids

i

and cholesterol groups

Consistent with partial expulsion of the peptide from the bilayer hydrophobic core, Fig.

16


Page 17 of 39

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6a shows that A β interacts almost exclusively with the cholesterol hydroxyl group GC1. Because this group can serve as a hydrogen bond donor, we computed the number of A β cholesterol hydrogen bonds

< Nhb,c (i) >

(Fig.

S7).

It is seen that, for the amino acids

Ile32-Leu34 and Lys28, the number of hydrogen bonds constitutes up to

≈20% of < Cc (i) >

underscoring hydrogen bond contribution to A β -cholesterol interactions. The contributions of DMPC lipids and cholesterol to hydrogen bonding with A β peptide are compared in Fig. S7. By applying the methodology described in Methods, we identied a single cholesterol binding site in the A β 10-40 monomer. As illustrated in Fig. 6b, it includes Phe19, Phe20, Lys28, and the continuous span of C-terminal residues Ile31-Val36, which together comprise only 29% of the A β sequence. Therefore, when cholesterol interacts with A β , it tends to crossbridge several residues distant along the sequence, which are from the central hydrophobic cluster R2, the turn region R3, and the hydrophobic C-terminal R4.

Indeed, the average

probability of observing such R2-R3, R2-R4, or R3-R4 cross-bridges for bound cholesterol molecules is 0.47.

On average, there are 3.3 ±0.4 cholesterol molecules bound to the A β

peptide, of which 2.5 ±0.3 interact with the binding site. Therefore, the binding site identied by us captures approximately 76% of all instances of cholesterol binding to A β . In summary, a single binding site distributed across the A β sequence serves as an anchor for cholesterol interactions with the peptide. The implications of this nding are analyzed in the Discussion.

Eect of A β binding on the bilayer structure According to our previous studies, A β peptide binding to the lipid bilayer disrupted its structure.

38

To probe the structural integrity of the DMPC bilayer with cholesterol, we

computed the number density of bilayer heavy atoms

r

to the Aβ center of mass and the distance

comparison, Fig. 7 also presents

nBL (r, z)

z

nBL (r, z)

as a function of the distance

to the bilayer midplane (Fig.

for the cholesterol-free DMPC bilayer.

7).

38

For

In the

gure we distinguish two bilayer regions: proximal, which is beneath the bound A β peptide,

17



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Page 18 of 39

(a)

(b)

Figure 6: (a) The number of contacts cholesterol structural groups

k.

< Cc (i, k) > formed < Cc (i, k) >

The value of

between A β amino acids

i

and

is color-coded according to the

scale on the right. Regions R1-R4 are colored following Fig. 1a. The gure demonstrates that Aβ predominately binds to the hydroxyl group GC1. (b) A snapshot of the A β peptide binding to the cholesterol-rich DMPC bilayer. The cholesterol binding site is composed of Ile31-Val36 in blue, Phe19 and Phe20 in green, and Lys28 in red.

Cholesterol molecule

interacting with this binding site is in purple.

and distant, unaected by A β . In the cholesterol-rich bilayer, the boundary between proximal and distant regions occurs at maximum.

Rc =11

Å, where the number of A β -bilayer contacts reaches

For the cholesterol-free DMPC bilayer,

cholesterol, the proximal region ( r

< Rc )

Rc =14

Å.

37

In the bilayer without

features a dramatic depletion of lipid density

18


Page 19 of 39

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beneath the bound A β peptide. In contrast, a less pronounced drop in

nBL (r, z) is observed in

the proximal region of the cholesterol-rich bilayer. To quantify density changes, we considered the bilayer heavy atom number densities,

nprox

and

ndist ,

over proximal and distant regions of the leaet ( 0 dened by the distant bilayer boundary

zb

computed by averaging

< z < zb ).

in Fig.

7.)

nBL (r, z)

(Note that a leaet was

We found that, from the distant

ndist =

to proximal regions, the cholesterol-rich bilayer number density decreases 19% from

0.037 ± 0.000Å−3

to

nprox = 0.030 ± 0.002Å−3 .

free DMPC bilayer is from

The corresponding change in the cholesterol-

ndist = 0.035±0.000Å−3

to

nprox = 0.024±0.003Å−3

or a decrease

of 31%. Therefore, consistent with visual inspection of Fig. 7, cholesterol noticeably reduces the bilayer density depletion beneath the bound A β peptide. In agreement with weak impact on the density of cholesterol-rich bilayer, A β binding does not aect the distribution of cholesterol molecules. To illustrate this, we compared their surface number densities

ns,c

in the distant and proximal regions identifying the positions of

cholesterol molecules with C13 atoms (Fig. 1c). We found that at 0.010 Å

−2

(with the errors of

±0.000

and

±0.001

Å

−2

ns,c

in both regions remain

, respectively). To further validate

this result, we computed the cholesterol-cholesterol radial density functions is the distance between two molecules. The functions

gCC (r)

gCC (r),

where

r

obtained in the proximal and

distant regions were found to be in a good agreement, indicating that, on average, in both regions a pair of nearest-neighbor cholesterol molecules are separated by the distance

rCC =6

Å. The impact of A β binding on the bilayer structure can be further investigated by computing the bilayer thinning previous studies. (r

51

∆D.

To this end, we followed the approach developed in our

Specically, we assumed that the bilayer boundary in the distant region

> Rc ) occurs at the constant z = zb dened from the condition nBL (r, zb ) = nw (r, zb ) ≡ nb ,

where

nw (r, z)

is the number density of water atoms. With this denition,

DMPC bilayer with cholesterol and 20 Å for the cholesterol-free DMPC. region, which can be dehydrated, we dene the

r-dependent

19


zb = 23

51

Å for the

In the proximal

bilayer boundary

zb (r)

using


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7: The number density to the Aβ center of mass

r

nBL (r, z)

Page 20 of 39

of bilayer heavy atoms as a function of the distance

and the distance

z

to the bilayer midplane.

The right panel

corresponds to the DMPC bilayer with cholesterol, whereas the left panel serves as a control describing the cholesterol-free DMPC bilayer.

38

nBL (r, z) is color-coded accordboundaries of the bilayers zb (r).

The value of

ing to the scale on the right. The solid lines indicate the

The vertical dashed lines separate proximal and distant regions. This gure demonstrates that cholesterol attenuates structural perturbations in the bilayer caused by A β .

the condition

nBL (r, zb (r)) = nb .

in the upper and lower leaets.

Then, the bilayer thickness

is the distance between

The change in bilayer thickness

estimated by taking the dierence in proximal region, i.e.,

D

D

∆D

zb (r)

or thinning can be

between the distant region and the center of the

∆D = D(r > Rc ) − D(r < 6

Å). We found that

the cholesterol-rich bilayer, whereas for the cholesterol-free bilayer

∆D =

7.3±2.0Å for

∆D =12.7±2.7

Å,

51

i.e.,

cholesterol reduces bilayer thinning by about 5 Å or 43%. If cholesterol decreases the bilayer thinning caused by A β binding, does it reduce the perturbations in DMPC lipids? deuterium order parameter

To check this possibility, we computed the lipid-carbon

< SCD (i) >

for carbons

i

in the proximal and distant

fatty acid tails. If fatty acid tails are disordered by A β , a dierence between

sn − 2

< SCD (i) >

computed for proximal and distant lipids will be evident. Fig. 8, which presents the average dierence

< ∆SCD (i) >, unexpectedly demonstrates that the extent of fatty acid disordering

in the cholesterol-rich and -free bilayers is about the same. Indeed, for the DMPC bilayer

20


Page 21 of 39

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with cholesterol, the average value of

− < ∆SCD >

across all carbons is 0.033 ±0.009,

whereas it is 0.032 ±0.002 for the pure DMPC bilayer.

In addition, the inset to Fig.

8

conrms a well-known result that cholesterol orders lipid fatty acid tails manifested in sharp increase of

− < SCD (i) > for all carbons i. 59

Taken together, these observations demonstrate

that cholesterol reduces bilayer thinning and lipid density depletion within the A β binding footprint, but surprisingly does not mitigate the disorder in the DMPC lipids. These ndings are rationalized in the Discussion.

puted for each carbon

< ∆SCD (i) > com< ∆SCD (i) >=< SCD (i)dist >

− < SCD (i)prox

are computed for distant and

Figure 8: The dierence in the lipid carbon-deuterium order parameters

i in sn − 2 fatty acid tails. Specically, >, where < SCD (i)dist > and < SCD (i)prox >

proximal lipids, respectively. The data in black and red correspond to the DMPC bilayer with and without cholesterol. The gure implies that cholesterol does not change the extent of disorder in fatty acid tails caused by A β binding. order parameter

(Inset) The lipid carbon-deuterium

− < SCD (i) > for sn − 2 fatty acid carbons i in the DMPC bilayer with and

without cholesterol (in black and red, respectively). The dashed and solid lines correspond to distant or proximal lipids. The vertical bars show simulation errors. The inset shows that cholesterol orders fatty acid tails.

21



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Page 22 of 39

Discussion Mechanism of A β binding Using isobaric-isothermal REMD simulations, we probed the equilibrium binding of A β 1040 monomers to the zwitterionic DMPC bilayer containing cholesterol.

As a control, we

used our previous REMD simulations, which studied binding of the same peptide to the cholesterol-free DMPC bilayer.

37,38

Our four main results, which elucidate the A β binding

mechanism to the cholesterol-rich bilayer, are as follows. First, addition of cholesterol repositions A β peptide in the DMPC bilayer by shifting the average locations of amino acids away from the bilayer core toward the headgroup region (Fig. 4). As a result, the probability of insertion into the hydrophobic core of the cholesterolrich bilayer decreases for all A β sequence regions and only the hydrophobic C-terminal still retains the inserted state. This repositioning of A β toward the bilayer surface is revealed by multiple lines of evidence, from the computation of the peptide heavy atom number density along the bilayer normal (in Fig. S6) to the comparative analysis of A β interactions with bilayer headgroups and fatty acid tails (in Fig. 5 and Table S4). Perhaps, the most direct argument supporting A β partial expulsion from the bilayer is provided by the number of inserted

Ni

and surface bound

Ns

the addition of cholesterol reduces 9.2±0.4

52

amino acids. Using

Ni

from 13.2±4.0

52

P (z; i)

in Fig. 4, we determine that

to 8.8±2.3, whereas

Ns

increases from

to 11.5±0.5, respectively. Thus, the net result of adding cholesterol to the DMPC

bilayer is that the fraction of amino acids inserted into the bilayer decreases from 43 to 28%, whereas the fraction of surface bound amino acids increases from 30 to 37%. Consequently, the Aβ peptide becomes partially expelled from the hydrophobic core of the cholesterol-rich bilayer adopting the state

E.

Conversely, in the absence of cholesterol, the peptide state can

be described as partially inserted into the hydrophobic core (the state the

I →E

I ).

An illustration of

transition as well as of the disruption of intrapeptide salt-bridges can be found

in the formation of strong electrostatic interactions between cationic amino acids (such as

22


Page 23 of 39

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Lys28) and DMPC headgroups. It is also worth noting that cholesterol increases the fraction of unbound A β heavy atoms from 28% for the cholesterol-free bilayer to 35%. Second, consistent with A β partial expulsion, cholesterol considerably alleviates the disruption in the bilayer structure caused by peptide binding.

Specically, compared to the

pure DMPC bilayer, cholesterol signicantly reduces bilayer thinning (almost in half or by more than 5 Å) and the depletion of bilayer density beneath the bound A β . Given these ndings one would expect that cholesterol mitigates the disorder in fatty acid tails within the proximal region. Surprisingly, we detected no appreciable changes in the extent of lipid disordering caused by A β between the bilayers with and without cholesterol. (Note that this argument refers to the change in lipid-carbon deuterium order parameter between proximal and distant regions rather than to its absolute values.) This outcome is due to the strikingly dierent impact produced by A β on the distributions of DMPC lipids and cholesterol. Indeed, Aβ binding does not aect the cholesterol surface number density within the bilayer proximal region nor its distribution. The likely reason for this observation is that cholesterol is positioned relatively deep in the hydrophobic core of the bilayer (Fig. S8), where it remains largely unperturbed by A β binding. In contrast, A β binding strongly impacts the surface number density of DMPC lipids,

ns,l .

Specically, between distant and proximal regions

is decreased 43% from 0.014 ±0.000 to 0.008 ±0.001 Å

−2

ns,l

. This drop in lipid density is even

larger than that observed in the cholesterol-free bilayer (from 0.016 ±0.000 to 0.011 ±0.002 Å

−2

or 31%), but is entirely masked in Fig. 7 by cholesterol, which largely sustains the total

bilayer density beneath the A β binding footprint. Therefore, we conclude that cholesterol has a protective eect on the bilayer integrity against the impact of A β binding. Third, due to partial A β expulsion and localization of cholesterol deep in the bilayer core, few Aβ amino acids interact directly with cholesterol molecules. Our analysis indicates that the Aβ peptide contains a single binding site distributed across the A β sequence, which involves six hydrophobic C-terminal amino acids (Ile31-Val36), Phe19 and Phe20 from the central hydrophobic cluster, and cationic Lys28 from the turn region. This binding site, on

23



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Page 24 of 39

average, captures about 76% of all A β -cholesterol interactions. Importantly, due to the positioning of Aβ and cholesterol in the bilayer (Fig. S8), their interactions are largely conned to the cholesterol headgroup GC1, which forms hydrogen bonds with the A β peptide. Fourth, addition of cholesterol to the DMPC bilayer dramatically reduces the helical propensity in the bound A β peptide (namely, in half from

0.39 ± 0.03

to

0.20 ± 0.04).

As a

result, binding from the water environment to the cholesterol-rich bilayer does not cause a profound redistribution of A β secondary structure as it occurs for the pure DMPC bilayer.

37

Indeed, binding to the cholesterol-rich bilayer increases the helix propensity merely by a factor of 1.7, whereas for the cholesterol-free bilayer the increase is more than three-fold.

37

Partial expulsion of the A β peptide from the hydrophobic core of the cholesterol-rich DMPC bilayer may change A β binding energetics. To assess this possibility, we decomposed the contacts between A β and the cholesterol-rich DMPC bilayer into four categories following four amino acid types:

apolar, polar, cationic, and anionic.

We found that 49% of all

Aβ -bilayer interactions are formed by apolar amino acids, followed by polar (38%) and cationic (10%) residues.

Anionic amino acids make a negligible contribution (3%).

For

comparison, when A β binds to the cholesterol-free DMPC bilayer, apolar and polar amino acids contribute almost equally to binding (46 and 45%), whereas cationic and anionic amino acids make minor contributions (5 and 4%). This analysis suggests that cholesterol causes subtle changes in binding energetics reducing the contribution of polar residues and increasing the contribution of positively charged amino acids. These ndings are consistent with the largely hydrophobic character of the cholesterol binding site in the A β peptide and strong electrostatic interactions between Lys28 and DMPC headgroups.

Why does cholesterol destabilize A β helical structure? Upon addition of cholesterol to the zwitterionic DMPC bilayer, the helical propensity in the bound Aβ monomer is dropped approximately in half. which cholesterol destabilizes helix structure?

Then, what is the mechanism by

To answer this question, we compared the

24


Page 25 of 39

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changes in helix propensity along the A β sequence (where

< H(i) >

< H(i) >0

and

< ∆H(i) >=< H(i) > − < H(i) >0

are the helix distributions in the presence and absence

of cholesterol) with the distribution of A β -cholesterol contacts Aβ sequence, the correlation coecient between

< ∆H(i) >

< Cc (i) >.

For the entire

< Cc (i) >

is -0.51. If this

and

computation is restricted to the amino acids interacting most frequently with cholesterol and featuring the most profound helix disruption (Ile31-Met35), the magnitude of correlation coecient increases to -0.75, indicating that cholesterol contacts and destabilization of helix are coupled. Consistent with this conclusion, Fig. S7 shows that the formation of hydrogen bonds between A β and cholesterol hydroxyl groups peaks at Ile32. Indeed, on average, the Aβ sequence region Ile31-Met35, which largely represents the cholesterol binding site and exhibits the largest decrease in helicity, establishes

≈50%

of all backbone hydrogen bonds

with cholesterol. Therefore, we surmise that helical structure in the bound A β peptide is mainly lost because of hydrogen bonding with cholesterol embedded in the lipid bilayer.

Why does the cholesterol-rich bilayer expel the A β peptide? It is imperative to investigate the mechanism of A β expulsion. It is well known that cholesterol reduces the disordering in fatty acid tails manifested in the increase in the order parameter

−SCD

(inset to Fig.

reduces the area per lipid

Al .

8).

59

Consequently, cholesterol condenses DMPC lipids and

To illustrate this eect, we computed

cedure of Edholm and Nagle for a two-component system. the area per cholesterol

x.

Prediction of

various

x.

Ac

Ac

and

is constant, whereas

Al

Al

x,

This procedure presumes that

requires several A β -free simulations of the DMPC bilayer with

Specically, we considered

At large

following the pro-

depends on the cholesterol molar fraction

x=0,

0.1, 0.2, 0.3, 0.4, 0.44, and 0.5. Each A β -free

bilayer provides an estimate of its total surface area

x/(1 − x).

60

Al

A(x),

which is plotted as a function of

a linear t is applicable according to

x A(x) = Al (x) + Ac (x), Nl 1−x 25


(1)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

where

Nl

Page 26 of 39

is the number of DMPC lipids. Because the concentration of cholesterol

corresponds to a linear regime, application of Eq. (1) yields with the results of Edholm and Nagle ( Ac =27 Å Eq. (1) that

Al (x = 0.44)=55.7 Å2 .

2

Ac ≈28 Å2 , which is in agreement

at 323K).

60

Furthermore, we found from

Al (x = 0)=65.1 Å2 , 38

Since

reduces the area per DMPC lipid by 9.4 Å

2

x = 0.44

we conclude that cholesterol

or about 15%.

Because cholesterol condenses DMPC lipids, it changes the lateral pressure in the bilayer. This circumstance may shift the balance of partially inserted

I

and expelled

E

Aβ states,

which are populated in cholesterol-free and -rich bilayers, respectively. To check if changes in lateral pressure can explain A β partial expulsion, we followed the formalism of Cantor compared the work

W

bilayers. During the

z

distance

performed along the

I →E

I →E

61

transition by cholesterol-free and -rich

transition, the cross-sectional area of the peptide

A(z)

at the

from the bilayer midplane is reduced within the hydrophobic core and increased

near the bilayer surface. Its change is then

∆A(z) = AE (z) − AI (z),

where

AE (z) and AI (z)

are the cross-sectional areas of partially expelled and inserted A β states. To compute

A(z) ≈ πRg (z)2 ,

we assumed that

Aβ heavy atoms at the distance for details). -rich (x

and

where

z

Rg (z)

is the two-dimensional radius of gyration of

from the bilayer midplane (see Supporting Information

To compute the lateral pressure proles in the cholesterol-free ( x

= 0.44)

bilayers,

implemented in NAMD

47

p0 (z)

A(z),

and

p(z),

= 0)

and

we used the approach of Lindahl and Edholm

62

and Aβ -free simulations of the DMPC bilayer. Then, the dierence

in the work performed by the bilayer along the

I→E Z

∆W = W − W0 = −NA

transition is

Lz

∆p(z)∆A(z)dz,

(2)

0

where

W

p0 (z), NA

and

W0

are the works done by cholesterol-rich and -free bilayers,

is Avogadro's number, and

Lz

is the periodic

z

∆p(z) = p(z) −

boundary of the system.

numerical integration in Eq. (2) was performed using the trapezoidal rule with We determined that

∆W = −1.5

The

∆z = 1Å.

kcal/mol. Importantly, this result is qualitatively robust

26


Page 27 of 39

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and does not depend on the details of calculations (see Supporting Information). In general, a positive work the

E

W

performed by the bilayer during the

state of a peptide by reducing its free energy by

I → E W.

transition would stabilize

However, because

∆W < 0,

it implies that, compared to the pure DMPC bilayer, cholesterol increases the free energy of the partially expelled state

E

of the Aβ peptide by 1.5 kcal/mol or

2.2RT .

Therefore,

changes in lateral pressure in the DMPC bilayer induced by cholesterol cannot rationalize A β partial expulsion. It must be noted in this context that lateral pressure is sensitive to bilayer composition and in the bilayers formed of dierent lipids, e.g., unsaturated ones, lateral pressure might still contribute to A β expulsion by switching the sign of

∆W . 63

We also note

that lateral pressure in the POPC bilayer may aect the aggregation of C99 monomers. The work between

I

W

and

64

performed by the bilayer does not account for a full free energy dierence

E

states. Therefore, there must be another mechanism of partial expulsion

of Aβ peptide from the cholesterol-rich bilayer unrelated to lateral pressure changes.

We

rst hypothesized that partial expulsion of the peptide is caused by its interactions with cholesterol. Specically, the A β peptide may shift up along the bilayer normal to maximize Aβ -cholesterol interactions. We ruled out this rationale, because the average changes in the positions of amino acids

i, ∆z(i), induced by cholesterol in Fig.

with the number of contacts with cholesterol

< Cc (i) >.

4 are only weakly correlated

Alternatively, a partial expulsion

of Aβ can be explained by the cholesterol-induced changes in the energy of non-bonded interactions between the peptide and the bilayer,

Eb (z) > as a function of the distance z

< Eb >.

To this end, we plot in Fig. S9

of the Aβ center of mass to the bilayer midplane. The

plot demonstrates that, for the DMPC bilayer with cholesterol, minimum at

z = 24Å,

has a pronounced

which is entirely absent in the cholesterol-free bilayer. Because in the

cholesterol-rich bilayer, the A β center of mass is located, on average, at

z = 26.1 ± 1.5Å, the

energy minimum at 24Å in Fig. S9 approximately reects the partially expelled state

E.

Furthermore, the average energy of interactions between A β and the cholesterol-free bilayer (in the state

I)

is

< Eb >≈ −246 ± 47

kcal/mol, whereas, in the presence of cholesterol (in

27



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the state

Page 28 of 39

E ), < Eb > is lowered by ≈ 33 kcal/mol to −279±34 kcal/mol.

Interestingly, in the

partially expelled state, the energy of A β interactions with DMPC lipids alone (excluding cholesterol) is

−252 ± 29

kcal/mol, which is still lower than

< Eb >

for the cholesterol-free

bilayer. This result is striking given that the number of DMPC lipids in the cholesterol-free system is one-third larger than in the bilayer with cholesterol.

Finally, to provide direct

evidence that, in the cholesterol-rich bilayer, the partially inserted state

I

is energetically

unstable, we seeded one of the REMD trajectories with initial structures, featuring the deeply inserted peptides. Specically, A β centers of mass were placed at the distance

z≈

5

Å from the bilayer midplane. At 330 K, both peptides in the upper and lower leaets became expelled to the

E

|z| >

20 Å within 4.7

ns

of REMD simulations, i.e., the system equilibrates to

state in less than 25% of the 330 K replica simulation time.

Taken together, our

analysis argues that dense packing of the DMPC lipids in the cholesterol-rich bilayer makes the partially expelled state

E

energetically favorable over the partially inserted state

I.

We

thus conclude that the partial expulsion of the peptide from the cholesterol-rich bilayer is driven by Aβ binding energetics rather than bilayer pressure changes.

Comparison with other studies The impact of cholesterol on A β binding to lipid bilayers has been the focus of several experimental studies. Using X-ray diraction experiments, it has been shown that the addition of cholesterol up to the molar fraction of

x = 0.4

into the weakly anionic DMPC:DMPS

(92:8 mol %) bilayer reduces deep embedding of A β 1-42 peptide in the bilayer core.

16

A

more quantitative picture of cholesterol's impact on the binding of the A β 25-35 fragment was provided by neutron diraction studies.

65

Those investigations have shown that the

increase in cholesterol molar fraction from 0.3 to 0.4 in the unsaturated POPC:POPS bilayer decreases the fraction of the embedded peptide from 45 to 30%, while concurrently increasing the population of surface-bound A β 25-35. These experimental observations are in a good qualitative agreement with our ndings suggesting that cholesterol partially expels

28


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Aβ 10-40 peptides from the core of cholesterol-rich DMPC bilayer. Interestingly, the ability of cholesterol to expel molecules embedded in the lipid bilayer is not limited to A β . In fact, X-ray diraction investigation of DMPC bilayers containing

x = 0.2

of cholesterol revealed

that it expels ibuprofen molecules from the bilayer core forcing them to reside on the bilayer surface.

66

It is instructive to compare the cholesterol binding sites identied in our and previous studies. A recent NMR study has probed the binding of cholesterol to the C-terminal fragment of the

β -amyloid

precursor protein, C99, which incorporates the entire A β sequence.

34

The analysis of chemical shifts in C99 embedded in micelles has indicated that cholesterol binds to the GXXXG motifs (G29-XXX-G33-XXX-G37) as well as to Glu22 and Asn27 in the Aβ region of C99. Subsequent all-atom molecular dynamics simulations of C99 spanning the cholesterol-rich DMPC bilayer have added anionic Asp23 to the list of amino acids governing cholesterol binding.

35

Molecular simulations studying the short A β fragment, A β 22-35,

have pointed to two specic amino acids, Val24 and Lys28, which are critical for cholesterol binding.

36

At the same time, relatively few studies have analyzed binding of cholesterol to

the full-length A β peptide. Using a combination of experimental measurements and docking simulations, Scala et al have identied the linear fragment 22-35 in the A β 1-40 sequence as the cholesterol binding domain.

67

In a general agreement with these ndings, our simu-

lations have revealed that six hydrophobic amino acids Ile31-Val36 in the C-terminal and Lys28 represent the main constituent of the cholesterol binding site. Nevertheless, our study does not support the involvement of the GXXXG motifs in binding nor the formation of cholesterol-A β interactions at the positions Glu22, Asp23, Val24, and Asn27. With respect to the GXXXG motif, this outcome is expected given low helical propensity of A β 10-40 bound to the cholesterol-rich DMPC bilayer. Additionally, our data suggest that aromatic amino acids Phe19 and Phe20 participate in the cholesterol binding site.

Therefore, the

mechanisms of cholesterol binding to A β 10-40 peptides and C99 or short A β fragments appear to be dierent reecting distinct positions and structures of these species in the lipid

29



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bilayers. Finally, we speculate on potential implications of our results for the mechanism of A β cytotoxicity. Partial expulsion of A β peptides from the core of DMPC bilayer, as observed in our simulations, may promote their aggregation on the surface of the lipid bilayer, which can act as an aggregation template.

32,33

On the other hand, our analysis strongly argues that

cholesterol protects the structural integrity of the lipid bilayer against perturbation caused by a bound Aβ monomer. Therefore, the cytotoxicity of A β peptides against cholesterolcontaining cellular membranes may result from a subtle interplay between these two factors. Either way, the REMD simulations reported in this study oer a molecular description of these two conicting factors, which promote or suppress A β cytotoxicity.

Conclusions In this study, we applied an all-atom explicit membrane and water model coupled with REMD simulations to investigate the equilibrium binding of A β 10-40 monomers to the zwitterionic DMPC bilayer containing cholesterol. Our previous REMD simulations, which studied binding of the same peptide to the cholesterol-free DMPC bilayer, served as a control against which we measured the impact of cholesterol. Our main objective was to elucidate the molecular mechanisms underlying the impact of cholesterol on A β binding to the bilayer. In summary, our conclusions are four-fold. First, addition of cholesterol to the DMPC bilayer partially expels the A β peptide from its hydrophobic core and promotes peptide binding to bilayer polar headgroups.

Using thermodynamic and energetics analyses, we argued that

Aβ partial expulsion is not related to cholesterol-induced changes in lateral pressure within the bilayer, but is caused by binding energetics, which favors A β binding to the surface of the densely packed cholesterol-rich bilayer. Second, cholesterol has a protective eect on the DMPC bilayer structure against perturbations caused by A β binding. In particular, cholesterol signicantly reduces bilayer thinning and overall depletion of bilayer density beneath

30


Page 30 of 39

Page 31 of 39

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the Aβ binding footprint. Third, the A β peptide contains a single cholesterol binding site distributed across the peptide sequence, which involves hydrophobic C-terminal amino acids (Ile31-Val36), Phe19 and Phe20 from the central hydrophobic cluster, and cationic Lys28 from the turn region. Together, this binding site comprises less than a third of all A β 10-40 amino acids but accounts for about 76% of all A β -cholesterol interactions.

Importantly,

the cholesterol binding site in the A β 10-40 peptide does not contain the GXXXG motif featured in cholesterol binding to the transmembrane domain C99 of the

β -amyloid

precur-

sor protein. This outcome suggests that the mechanisms of cholesterol binding to A β and C99 are distinct reecting their dierent conformations and positions in the lipid bilayer. Fourth, cholesterol present in the DMPC bilayer sharply reduces the helical propensity in the bound Aβ peptide. As a result, cholesterol largely eliminates a dramatic acquisition of helical structure observed upon A β binding from aqueous environment to the cholesterolfree DMPC bilayer.

We explain this eect by the formation of hydrogen bonds between

cholesterol and the A β backbone, which prevent helix formation. Comparison with available experimental data probing the impact of cholesterol on A β -bilayer interactions revealed a good agreement with our ndings. Hence, we expect that our simulations will advance understanding of the molecular-level mechanism behind the role of cholesterol in Alzheimer's disease.

Supporting Information: Further details concerning the model and methods used and addi-

tional data are provided in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding author:

∗

E-mail: [email protected]. Phone: 703-993-8395.

Author contributions: Both authors contributed equally to this work.

Notes: The authors declare no conict of interest.

31



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Page 32 of 39

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β

Amyloid

Cholesterol Changes the Mechanisms of AÎ² Peptide Binding to the

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