BINDING OF CYTOTOXIC AÎ²25-35 PEPTIDE TO THE DMPC LIPID

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BINDING OF CYTOTOXIC A#25-35 PEPTIDE TO THE DMPC LIPID BILAYER Amy K Smith, and Dmitri K. Klimov J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00045 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Journal of Chemical Information and Modeling

BINDING OF CYTOTOXIC Aβ 25-35 PEPTIDE TO THE DMPC LIPID BILAYER Amy K. Smith and Dmitri K. Klimov

∗

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

E-mail: [email protected]

Abstract Aβ 25-35 is a short, cytotoxic, and naturally-occurring fragment of the Alzheimer's Aβ peptide. To map the molecular mechanism of Aβ 25-35 binding to the zwitterionic DMPC bilayer, we have performed replica exchange with solute tempering molecular dynamics simulations using all-atom explicit membrane and water models. Consequences of sequence truncation on the binding mechanism have been measured by utilizing as a control our previous simulations probing binding of the longer peptide Aβ 10-40 to the same bilayer. The most intriguing feature of Aβ 25-35 binding to the DMPC bilayer is a coexistence of two bound states with strikingly dierent characteristics: a dominant surface-bound state and a less stable inserted state. In the surface-bound state, the peptide samples extended conformations, in which its unbound C-terminal is pointed away from the bilayer. In contrast, in the inserted state, the C-terminal resides deep in the bilayer hydrophobic core. In both states, the N-terminal remains anchored to the bilayer. Free energy landscape analysis reveals that the two states are separated by a moderate barrier, suggesting that Aβ 25-35 monomer may frequently interconvert between them. The net eect of Aβ 25-35 binding is a minor impact on the bilayer structure, which contrasts with the considerable bilayer perturbations induced by a longer Aβ 10-40 peptide penetrating deep into the bilayer core. Therefore, we conclude 1

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that the binding mechanisms of Aβ 25-35 and Aβ 10-40 peptides are dierent. Potential implications of our results for Aβ 25-35 cytotoxicity are discussed. A comparison of experimental data with our ndings reveals a good agreement.

Introduction Alzheimer's disease (AD) is a neurodegenerative condition leading to memory impairment and loss of cognitive function. A dening AD physical characteristic is the presence of extracellular senile plaques composed of amyloid-β (Aβ ) peptides aggregated into insoluble amyloid brils. Aβ derives from the amyloid precursor protein (APP), a much larger transmembrane protein expressed in all tissues, including the central nervous system. by

β-

and

γ -secretases

1

Proteolysis

cleaves Aβ from APP, releasing it into the extracellular space. The

predominant Aβ species found in senile plaques are 40 or 42 amino acids peptides, Aβ 1-40 and Aβ 1-42, with the former representing uid.

2,3

∼90% of the Aβ

species occurring in cerebrospinal

Numerous studies have rmly established that Aβ 1-40 and Aβ 1-42 peptides are am-

phiphilic, highly amyloidogenic, cytotoxic in vitro and neurotoxic in vivo capable of eroding intercellular synapses. cytotoxic forms.

47

It is currently thought that small soluble Aβ oligomers are the most

8,9

Besides the full-length alloforms Aβ 1-40 and Aβ 1-42, many shorter fragments of these peptides with dierent N- and C-terminal truncations have been detected and studied.

10

Some of the truncated species have been found to exhibit the same or even higher aggregative and/or cytotoxic properties than the full-length peptide.

11

One such truncated Aβ peptide

is the undecapeptide Aβ 25-35 shown in Fig. 1a, which is apparently the shortest naturallyoccurring fragment that retains some of the properties of the full-length peptide.

12

Indeed,

experimental studies have shown that Aβ 25-35 is amphiphilic, aggregative, amyloidogenic, and neurotoxic.

11,13,14

An interesting characteristic of Aβ 25-35 setting it apart from the

full-length Aβ is its immediate, without aging cytotoxicity in vitro.

12

This may suggest a

cytotoxicity of the monomer itself or a faster aggregation to cytotoxic species compared to

2


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the full-length peptide. Interestingly, experimental data support both possibilities. In particular, monomeric Aβ 25-35 is found to generate apoptotic signals causing cellular death.

11

At the same time, fresh Aβ 25-35 exhibits sedimentation indicative of aggregation within an hour, which is even more rapid than the sedimentation of Aβ 1-42.

4

Furthermore, the

sequence segment forming Aβ 25-35 is a biologically active region of the full-length peptide and includes APP extracellular (25-28) and transmembrane (29-35) residues.

12

As a result

many experimental and in silico studies have used Aβ 25-35 as a proxy for the full-length peptides.

11,1518

Aβ peptide interactions with cellular membranes have been widely studied in recent years.

1924

Experiments have shown that a full length Aβ 1-40 readily binds to lipid mem-

branes in vitro as monomeric or small oligomeric species.

23

Although the molecular mech-

anism of Aβ -lipid interactions awaits full understanding, experiments have demonstrated that Aβ can perturb cellular membranes presumably by forming structured pores or by destabilizing lipid-lipid interactions suciently to induce uncontrollable ion ux.

22,2529

In

line with these notions, electron density analysis of X-ray diraction patterns has revealed deep penetration of Aβ peptides into the bilayer hydrophobic core, potentially compromising its structural integrity.

30,31

Along with other Aβ species, Aβ 25-35 has been studied with

respect to membrane interactions, although the results have been somewhat controversial. For instance, CD and calorimetry experiments have suggested that Aβ 25-35 binding is limited to anionic lipid bilayers.

32

Later neutron diraction experiments have revealed Aβ 25-35

interactions with zwitterionic POPC membranes and suggested that the depth of peptide penetration depends on the lipid charge.

33

It is noteworthy that the depth of Aβ 25-35 pene-

tration into the bilayer may have direct implications for cytotoxicity. For example, a mixed experimental and simulation study of Aβ 25-35 and its mutants have shown that the deeperinserting mutants are non-toxic, while the shallowly inserted wild-type Aβ 25-35 is cytotoxic in vitro.

14

All-atom molecular dynamics simulations can provide important insights into molecular

3



Page 4 of 40

(a) Y10 E V H H Q K L V F F A E D V G25 S N K

(b)

G A I I G L M35 V G G V V40 L4

L1 – L3 L5

(c)

zp

0

-zp

Figure 1:

(a) The sequence of Aβ 25-35 peptide with the highlighted N-terminal R3 (in

blue) and C-terminal R4 (in red) regions. For reference, the sequence of the longer Aβ 10-40 peptide is shown in grey. (b) A DMPC lipid is divided into ve structural groups: choline (L1), phosphate (L2) with phosphorus P atom presented as sphere, glycerol (L3), and two fatty acid tails (L4 and L5). L1-L3 make up the polar lipid headgroups. The hydrophobic core is composed of L4 and L5. (c) A snapshot of DMPC bilayer with two bound Aβ 25-35 peptides. Lipids are shown in grey with phosphorus atoms distinguished as tan spheres. R3 and R4 regions in Aβ 25-35 peptides are in blue and red, respectively. Water is shown in light blue. The centers of mass of phosphorus atoms in each leaet are restrained, on average, at

±zP (zP = 17.37Å). mechanisms governing Aβ cytotoxicity. Recently, using replica exchange molecular dynamics (REMD) and all-atom explicit water and membrane models we have investigated equilibrium binding of Aβ 10-40 peptides to a zwitterionic DMPC bilayer.

3436

Those studies have estab-

lished that binding to the DMPC bilayer changes the conformational ensemble of Aβ 10-40 by

4


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triggering the formation of stable helix in the C-terminal, which is not observed in water.

37

More importantly, REMD simulations have found that two sections of Aβ sequence, the central hydrophobic cluster (CHC, 17-21) and the C-terminal (29-40), penetrate deep into the core of the DMPC bilayer, causing its thinning and structural disorder. Interestingly, the Aβ 10-40 polar turn region (22-28) situated between the CHC and C-terminal remains bound to the bilayer surface. Because Aβ 25-35 comprises parts of the turn region and the C-terminal of Aβ 10-40, our previous studies can serve as controls to investigate the impact of sequence truncations on Aβ -bilayer interactions. Thus, expanding and capitalizing on our earlier investigations, this paper applies replica exchange simulations to examine binding of Aβ 25-35 monomers to the zwitterionic DMPC bilayer (Fig. 1). Our primary goals are to study the distribution of conformational states that Aβ 25-35 adopts upon binding to the DMPC bilayer, to investigate Aβ 25-35 interactions with DMPC lipids, and to map the extent of bilayer structural disorder caused by Aβ 25-35. We use the results collected alongside this investigation to propose a molecular mechanism of Aβ 25-35 binding to zwitterionic bilayers.

Methods Molecular model and simulation system:

Our simulations probed the binding of

Aβ 25-35 peptides to the bilayer formed by dimyristoyl phosphatidylcholine (DMPC) lipids (Fig. 1). Peptides and lipids were modeled using the all-atom CHARMM22 force eld with CMAP corrections

38

and the all-atom CHARMM36 force eld,

39

respectively. Water was

represented with the modied TIP3P model. The peptide N- and C-terminals were capped with neutral acetylated and amidated groups, respectively. Two Aβ25

− 35

monomers were

placed on opposite sides of a 98-lipid DMPC bilayer (Fig. 1c). Each bilayer leaet consisted of 49 DMPC molecules arranged in a square shape in the x-y plane with the bilayer normal aligned along the z-axis. The design, in which two peptides bind to the opposite sides of the bilayer, prevents the development of bilayer curvature, which may occur if only one Aβ 25-

5



35 peptide binds to the bilayer.

40

Page 6 of 40

Furthermore, such symmetrical design of the simulation

system allowed us to probe simultaneous binding of two Aβ 25-35 monomers to the DMPC bilayer. The peptides and bilayer were solvated with 4330 water molecules, and two chloride counterions were added to neutralize the system's net charge. The total number of atoms was 24,878, and the initial dimensions were 56.0Å x 56.0Å x 77.8Å. A control simulation system was designed to probe conformational properties of Aβ 25To this end, Aβ25

35 in lipid-free water.

− 35

monomer was solvated with 1602 TIP3P

water molecules and one chloride counterion for a total of 4968 atoms in a cubic box. Initial dimensions were 36.7Å x 36.7Å x 36.7Å. As a further control we used our previous REMD simulations probing binding of a longer Aβ 10-40 peptide to the same DMPC bilayer.

34,35

The use of two control systems permits us to evaluate the eect of binding to the DMPC bilayer on the peptide's conformation and the consequences of sequence truncation.

Replica exchange simulations:

To enhance conformational sampling, we performed

isobaric-isothermal (NPT) replica exchange with solute tempering (REST) molecular dynamics simulations.

41

Following traditional replica exchange molecular dynamics (REMD),

REST replicates the molecular system peratures.

R

times and simulates it in parallel at

R

unique tem-

In contrast to REMD, REST replicas utilize unique scaling factors applied to

solute-solvent and solvent-solvent energies. Specically, using the scaling factors proposed by Camilloni et al,

42

the enthalpy

Hr

of the NPT system with pressure

s Hr = Ep +

where

Ep , Epw ,

and

Ew

energies, respectively. (0

≤ r ≤ R − 1).

and volume

V

β0 β0 Epw + Ew + P V, βr βr

is

(1)

are the solute-solute, solute-solvent, and solvent-solvent potential

The factor

Importantly, for

is recovered in Eq.

P

βr = (kB Tr )−1 r=0

corresponds to replica temperature

Tr

unperturbed Hamiltonian referred to as wild-type

(1), whereas in all other replicas (r

eectively keeps solvent temperature xed at

T0

6

> 0)

energy scaling in Eq.

(1)

without aecting the temperature of solute.


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It is noteworthy that the REST version based on Eq. (1) implements the same algorithm as designed by Berne and coworkers,

41

but it deviates from the original by scaling solvent-

solvent energies and using dierent temperatures for dierent replicas. Exchanges of system coordinates and velocities, attempted every 2

ps,

with the probability

Xr

and

Xr+1 ,

between neighboring replicas

r

and

r+1

were

and acceptance of exchanges follows from the Metropolis criterion

ω = min[1, e−∆ ],

where

∆

is

∆ = βr (Hr (Xr+1 ) − Hr (Xr )) + βr+1 (Hr+1 (Xr ) − Hr+1 (Xr+1 )).

As explained elsewhere,

36,41

(2)

the scaling factors employed in Eq. (1) eliminate solvent-solvent

energy contributions to the exchange probability

ω.

In practical terms, this drastically

lowers the number of degrees of freedom governing replica exchanges, broadens the energy distributions and, consequently, considerably reduces the number of replicas without aecting the temperature range or exchange rates. In our REST system

R=6

replicas were distributed exponentially in the temperature

range from 330 to 440K. We used slightly elevated temperature for the wild-type replica to facilitate conformational sampling and comparisons with the control Aβ 10-40 simulations. Aβ 25-35 peptides and two chloride counterions were designated as solute, and the bilayer and water were treated as a cold solvent. (Note that traditional REMD simulates lipids at dierent temperatures.

However, in the previous study we have compared REST and

REMD samplings of a peptide binding to the bilayer and found their distributions of states to be in perfect agreement.

36

) Temperature was controlled via underdamped Langevin dy-

namics with the damping coecient

γ = 5ps−1 ,

and the pressure was set to 1

atm

using the

Nose-Hoover Langevin piston method with piston period and decay of 200 and 100

f s,

spectively. For pressure control a semi-isotropic coupling scheme was utilized, in which

y

dimensions of the system were coupled, and the

z

x and

dimension was adjusted independently.

Periodic boundary conditions and an integration timestep of 1

7

re-


f s were used.

Covalent bonds


Page 8 of 40

associated with hydrogen atoms were constrained by the SHAKE algorithm. Electrostatic interactions were computed with Ewald summation, and van der Waals interactions were smoothly switched o from 8 to 12 Å. To prevent escape of lipid molecules from the bilayer across all replicas, we followed our previous studies

34

and introduced a harmonic restraint on the center of mass of phosphorus

atoms in a leaet with the force constant approximately xed the

z

k=

2 6.5 kcal/mol/Å . This restraining potential

coordinate of phosphorus atoms center of mass to that computed

at 330K for the restraint-free DMPC bilayer.

34

Applying the restraints to the center of mass

and not to individual phosphorus atoms preserves membrane exibility and the magnitude of their uctuations observed during the restraint-free simulations. A second set of restraints was utilized at the upper and lower

z

periodic boundaries to prevent the Aβ 25-35 peptides

from aggregating. Specically, a repulsive harmonic potential with the force constant kcal/mol/Å either

z

2

was applied to the

z

k = 10

coordinates of Aβ 25-35 atoms occurring within 4 Å from

boundary. Impact of bilayer restraints is discussed in Supporting Information.

For the control lipid-free water system, we used

R = 4

REST replicas distributed ex-

ponentially in the range from 330 to 410K. Aβ 25-35 peptide and chloride counterion were assigned as solute, and the water was treated as solvent. No restraints were applied to this system, and the pressure control used the scheme in which uctuations of the unit cell dimensions were isotropically coupled. All other modeling methodologies were the same as for the DMPC system. Simulations were performed using the program NAMD

43

and in-house scripts for managing replicas and exchanges.

with REST implementation

44

For the DMPC system, the

preliminary REST simulations consisting of six trajectories and initiated with extended, unbound Aβ 25-35 structures collected 40 ns of sampling per replica in a trajectory.

The

objective of these simulations was to generate a diverse pool of inserted and surface-bound peptide states. These states were then used to initiate six production REST trajectories. To minimize bias from initial conditions, each production REST trajectory started with unique

8


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and equal mixture of inserted and surface-bound states. During production simulations each replica in a trajectory was sampled for 20

ns.

System equilibration in the preliminary and

production REST simulations was monitored using two probes, the probability distribution

P (zcom )

of the

z

position of Aβ 25-35 center of mass

the helix propensities

hH(i)i

for amino acids

i.

zcom

along the bilayer normal and

Consequently, initial 42 ns of sampling

per replica in a trajectory (thus spanning the entire preliminary simulations and 2 ns of production) were discarded as non-equilibrium, whereas the remaining 18 ns of sampling per replica in a trajectory were considered as equilibrated. The total equilibrium sampling across all replicas and trajectories is then 0.65

µs.

Because Aβ 25-35 peptides bind to the

DMPC bilayer independently (see below), the equilibrium sampling per peptide is eectively doubled to 1.3

µs.

The average exchange rate between replicas was 25%.

The REST simulations in lipid-free water were initiated with Aβ 25-35 random coil conformations. In all, eight independent REST trajectories were produced collecting 0.64

µs

of

sampling. After discarding unequilibrated portions in each trajectory, the equilibrium sampling was reduced to 0.58 at 32%.

µs.

The average exchange rate between replicas was maintained

Full analysis of REST performance, equilibration, and convergence is presented

in the Supporting Information.

To compute sampling errors in the wild-type replica, we

considered each of REST trajectories as an independent sample and used standard block averaging technique.

Computation of structural probes: tide,

34

Following our previous studies of Aβ10

− 40

pep-

we divide Aβ 25-35 peptide into the polar N-terminal R3 (residues 25-28) and the

hydrophobic C-terminal R4 (residues 29-35) as shown in Fig. 1a. The DMPC molecule is divided into ve structural groups as shown in Fig. 1b. Intra- and intermolecular interactions were determined by considering the positions of centers of mass of lipid structural groups and amino acid side chains. A contact between them is formed when the distance between their centers of mass is less than 6.5 Å. This threshold distance approximately corresponds to the onset of side chain hydration as the distance between them increases. Aβ 25-35 pep-

9



Page 10 of 40

tide is considered bound to the bilayer if at least one amino acid side chain forms a contact with any lipid group. Using the contact denition, we checked that Aβ 25-35 peptides do not interact between themselves or with their periodic images. Aβ 25-35 secondary structure was assigned using the program STRIDE,

45

and

α-, 310 -,

or

π

helices were pooled into a helical

state. Aβ 25-35 localization in the DMPC bilayer was described using several probability distributions. First, the probability

P (z; i)

for an amino acid

i

to occur at a distance

the bilayer midplane was computed. The insertion probability as the probability for phorus atoms amino acid Aβ25

− 35

i

zP .

i

z

from

Pi (i) was derived from P (z; i)

to reside below the average position of the center of mass of phos-

Similarly, the surface binding probability

to reside in the lipid headgroup region (zp

Ps (i)

is the probability for an

< z < zP + 6.5

Å). The impact of

peptide on the DMPC bilayer structure was probed with several measures. The

number density

nl (r, z) of DMPC heavy atoms with respect to the distance r to the Aβ 25-35

center of mass and the distance

z

to the bilayer midplane was computed, thus mapping a

cross-sectional distribution of lipid atoms in the bilayer. The lipid carbon-deuterium order parameter

SCD

was determined for carbons 2 through 14 in the

sn − 2 fatty acid tails dened

as

SCD = where

θ

3cos2 θ − 1 , 2

(3)

is the angle between the bilayer normal and the C-H bonds in the fatty acid tail.

The fatty acid tails were also characterized by their tilt angles

γ

dened as the angle between

the bilayer normal and the vector formed between the rst and last carbons in the tail. For example,

γ = 180◦

corresponds to a fatty acid tail aligned with the bilayer normal without

a tilt. All structural probes are reported as their thermodynamic averages computed for the unscaled, wild-type replica

r=0

at 330K. The quantities are further averaged over the two

peptides or bilayer leaets.

Conformational clustering: VMD,

46

Clustering of peptide conformations was performed using

which implements the method of Daura et al.

10

47

For each pair of peptide structures we


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have computed the RMSD between backbone Cα atoms after peptide alignment, and clusters were then dened by the RMSD cuto value

R0 .

To select appropriate

R0 we analyzed cluster

populations at various RMSD cutos and monitored the number of populated clusters, each of which must represent at least 10% of all structures and, together they must capture at least 50% of all structures in Aβ 25-35 conformational ensemble. If the latter condition is satised, we have selected as

R0

the largest RMSD cuto that occurs prior to a major

reorganization of clusters, e.g., when two smaller distinct populated clusters merge into one larger cluster.

The clusters are assumed distinct if they exhibit dierent distributions of

secondary structure, peptide dimensions, or positions in the bilayer. Structural properties of a cluster are reported as averages over all conformations assigned to a cluster.

Results Structure of Aβ 25-35 peptide bound to DMPC bilayer We rst examine the secondary structure of Aβ 25-35 monomer bound to the zwitterionic DMPC membrane. In the bound state, which occurs with the overwhelming probability of 0.94 at 330K, Aβ 25-35 adopts helix, turn, and random coil conformations with almost equal propensities (hHi

= 0.31 ± 0.03, hT i = 0.36 ± 0.02,

and

hRCi = 0.33 ± 0.02,

For comparison, the Aβ 25-35 helical propensity in lipid-free water is

respectively).

hHi = 0.15 ± 0.03

indicating that binding to the DMPC bilayer increases the helix population about two-fold. The helix propensities

hH(i)i

for amino acids

i

are presented in Fig. 2. The gure reveals

that in water environment the helix propensity is uniformly low across the entire sequence (hH(i)i

< 0.25).

However, binding to the DMPC bilayer increases

hH(i)i for all amino acids,

particularly in the R4 region. Indeed, when Aβ 25-35 is bound, three amino acids, Ile32, and Gly33, adopt stable helix with

hH(i)i0.50.

i=Ile31,

Therefore, binding to the DMPC

bilayer reorganizes Aβ 25-35 secondary structure, with most of the change occurring in the hydrophobic C-terminal.

11



Figure 2:

Helix propensities

hH(i)i

for Aβ amino acids

i.

Page 12 of 40

The data in black and blue

correspond to the Aβ 25-35 peptide bound to the DMPC bilayer or solvated in lipid-free water, respectively. The data in red represent Aβ 10-40 peptide. NMR structure.

34

48

hH(i)i for the residues 25-35 within the longer

The light blue shaded area marks the helix region in the experimental Sampling errors are shown by vertical bars.

Regions R3 and R4 are

colored following Fig. 1a. The plot shows that binding to the DMPC bilayer promotes helix propensity in Aβ 25-35, but to a much smaller extent than in the longer peptide Aβ 10-40.

Fig. 2 also presents the helix propensities for the residues 25-35 within the longer Aβ 1040 peptide (denoted as Aβ 25-35(10-40)), which binds to the same DMPC membrane.

34

Within Aβ 25-35(10-40) there are two regions featuring a stable helix, Gly25-Ser26 and, particularly, Ile31-Met35, where helix fraction reaches

hH(i)i > 0.80.

Consequently, the total Aβ 25-35(10-40)

hHi = 0.59 ± 0.06 exceeding that of Aβ 25-35 almost two-fold.

feature setting Aβ 25-35 apart from Aβ 25-35(10-40) is that

hH(i)i

Another

varies smoothly along

Aβ 25-35 sequence, whereas Aβ 25-35(10-40) exhibits a distinct gap in helix structure at Asn27 and Lys28. The likely reason is that truncation eliminates Asp23-Lys28 salt bridge and several hydrophobic contacts between the central hydrophobic cluster and C-terminal allowing moderate secondary structure changes at

i =Asn27

and Lys28.

49

Thus, Aβ 25-35

peptide and the corresponding region in Aβ 10-40 adopt helical conformations upon binding to the DMPC bilayer, but this eect is far more pronounced in the longer peptide than for Aβ 25-35.

12


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To probe Aβ 25-35 tertiary structure, we computed its contact map

hC(i, j)i,

ports the probabilities of forming intrapeptide contacts between amino acids average, Aβ 25-35 forms

i

which re-

and

j.

On

hCi = 7.2±0.1 intrapeptide contacts, of which only two, Gly29-Ile32

and Gly29-Gly33, are stable, i.e., their

hC(i, j)i > 0.5.

These two local contacts reside in

the R4 region reecting the formation of helical structure in the bound Aβ 25-35. To perform quantitative comparison of the tertiary interactions in Aβ 25-35 and Aβ 25-35(10-40), we dened the dierence contact map

h∆C(i, j)i = hC(i, j)i25−35 − hC(i, j)i10−40 .

It follows

that the contact most aected by truncation is Ser26-Lys28 in the R3 region, which is highly stable within Aβ 25-35(10-40) (hC(26, 28)i (hC(26, 28)i

= 0.33±0.04).

= 0.76±0.08), but is largely disrupted in Aβ 25-35

We show below that the disruption of this contact contributes to

strong binding of the respective amino acids to the DMPC bilayer. Overall, Aβ 25-35(10-40) forms slightly fewer contacts than Aβ 25-35 (hCi

= 6.6 ± 0.4).

Binding of Aβ 25-35 to the DMPC bilayer Because the conformational ensembles of bound Aβ 25-35 and Aβ 25-35(10-40) are distinct, one may expect that the respective peptides interact dierently with the DMPC bilayer. To check this expectation, we computed the probabilities occur at the distance

z

P (z; i)

for Aβ 25-35 amino acids

i

to

from the bilayer midplane (Fig. 3a). Using these probabilities we

can determine the average location

hz(i)i of each amino acid i along the bilayer normal.

Fig.

3a shows that all Aβ 25-35 amino acids reside, on average, well above the DMPC phosphorus atoms, i.e.,

hz(i)i > zP .

Therefore, Aβ 25-35 amino acids must be typically surface-bound to

the DMPC bilayer or remain solvated. This nding contrasts sharply with the distribution

hz(i)i

for Aβ 25-35(10-40), which demonstrates penetration of four R4 residues (Ile31, Ile32,

Gly33, Met35) into the bilayer hydrophobic core below To gain better insight, we have used of binding to the bilayer surface,

i

(see Methods).

zP . 50

P (z; i) to compute the probabilities of insertion, Pi (i),

Ps (i), and of remaining solvated, Pu (i), for each amino acid

The distributions of these probabilities along Aβ 25-35 sequence in Fig.

13



Page 14 of 40

3b implicate dierent behaviors of N- and C-terminals. The average probability of surface binding for the N-terminal R3 residues R4 (Ps (R4)

= 0.17 ± 0.03).

with the probability almost 50% to

Pi (R3) = 0.20 ± 0.02,

Pi (i) + Ps (i),

terminal (0.66 ± 0.01 vs unbinding (Pu (i)

whereas for R4 residue this probability increases

Furthermore, the overall probability of interaction

is clearly enhanced in the N-terminal compared to the C-

0.46 ± 0.03, respectively).

> 0.50)

is considerably larger than for

It also follows from Fig. 3b that the typical R3 residue inserts

Pi (R4) = 0.29 ± 0.01.

with the bilayer,

Ps (R3) = 0.46 ± 0.03

Consequently, the largest probabilities for

are observed in the C-terminal R4. Therefore, the N-terminal R3

is typically bound to the DMPC bilayer, whereas the C-terminal R4 has a weak preference for being unbound. Using the probabilities in Fig. 3b it is straightforward to compute the average numbers of Aβ 25-35 amino acids classied as inserted unbound

hNu i

acids (hNu i

hNi i,

surface bound

hNs i,

or

(Table 1). It follows from the table that almost half of 11 Aβ 25-35 amino

= 5.2 ± 1.0

or 47%) remains unbound, whereas approximately equal fractions

are either inserted (25%) or surface bound (27%). Aβ 25-35 binding diers sharply from that of Aβ 25-35(10-40). According to thermodynamic data given in Table 1, out of 11 Aβ 25-35(10-40) residues, 53% are inserted and 21% are surface-bound, leaving only 26% unbound. Therefore, more than half of Aβ 25-35(10-40) amino acids are typically inserted into the bilayer core, in contrast to Aβ 25-35 peptide, for which this fraction is more than twice smaller. Furthermore, the fraction of unbound Aβ 2535 amino acids is almost two-fold larger than the corresponding fraction for Aβ 25-35(10-40). Table 1: Amino acid categories observed upon Aβ interaction with the DMPC bilayer

System or state Aβ 25-35 Aβ 25-35 (state S) Aβ 25-35 (state I) Aβ 25-35(10-40)a

hNi i 2.8 ± 0.7 0.5 ± 0.1 8.4 ± 0.1 5.8 ± 1.9

hNs i 3.0 ± 0.6 3.2 ± 0.2 2.3 ± 0.1 2.3 ± 1.3

a

Data for Aβ 25-35(10-40) are from previous study 34

b

hNi i + hNs i

hNi i + hNs ib 5.8 ± 1.2 3.7 ± 0.3 10.7 ± 0.2 8.1 ± 2.1

hNu i 5.2 ± 1.0 7.3 ± 0.2 0.3 ± 0.0 2.9 ± 2.1

gives the total number of amino acids interacting with the bilayer

To directly probe Aβ 25-35 interactions with the DMPC bilayer, Fig.

14


4 presents the

Page 15 of 40 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


(a)

(b)

Figure 3: (a) The probability distributions the distance the scale.

z

from the bilayer midplane.

P (z; i)

for Aβ 25-35 amino acids

The average positions of Aβ 25-35 amino acids

i hz(i)i

to occur at

are given by black line,

whereas the red line presents the same quantity for Aβ 25-35(10-40).

z = zP

i

The probabilities are color coded according to

50

The dashed line at

separates the bilayer hydrophobic core and polar surface. (b) The probabilities of

insertion

Pi (i)

(in red), of binding to the bilayer surface

solvated in water phase

Pu (i)

Pi (i) + Ps (i)(= 1 − Pu (i))

Ps (i)

(in green), and to remain

(in blue) are shown for Aβ 25-35 amino acids

i.

The sum

gives the probability of interaction with the bilayer presented by

black line. Sampling errors are shown by vertical bars. Some errors in (b) are omitted for clarity. Regions R3 and R4 are colored following Fig. 1a. The panels show that Aβ 25-35 binding to the DMPC bilayer is not uniform along the sequence - the N-terminal R3 anchors binding to the bilayer, whereas the C-terminal R4 alternates between unbound (preferred) or inserted states.

average numbers of contacts between amino acids

i

and lipids,

hCl (i)i.

Consistent with the

analysis of Aβ 25-35 localization along the bilayer normal, the residues forming the largest numbers of interactions with the DMPC bilayer are found in the Aβ 25-35 N-terminal Ser26 (hCl (26)i

= 1.8 ± 0.1),

Gly25 (1.6

± 0.2), 15

Lys28 (1.6


± 0.2),

Asn27 (1.4

± 0.1)

-


and

hCl (i)i

Page 16 of 40

generally decreases toward the C-terminal. Although Aβ 25-35 and Aβ 25-35(10-

40) form about the same number of contacts with the bilayer (Table S1 in Supporting Information), their distributions along the sequence dier.

To compare them, we dene

the residue-specic dierence in the number of lipid contacts between Aβ 25-35 and Aβ 2535(10-40)

h∆Cl (i)i

=

hCl (i)i25−35 − hCl (i)i10−40 .

Its plot in Fig.

4 reveals that Aβ 25-35

lipid contacts formed by Ser26 and especially Lys28 are strongly enhanced, whereas the contacts along the hydrophobic C-terminal are suppressed compared to Aβ 25-35(10-40). These ndings point to dierences in binding energetics. To further probe this aspect, Table S1 from Supporting Information lists the total numbers of lipid contacts for both peptides categorizing them with respect to polar, apolar, and cationic amino acids.

Although in

both peptides apolar amino acids make the largest contribution to binding, the table shows that, in comparison to Aβ 25-35(10-40), Aβ 25-35 features more polar and especially cationic interactions at the expense of apolar contacts. Furthermore, out of all contacts occurring between Aβ 25-35 and the bilayer (see Table S1), DMPC polar headgroups L1-L3, while only

9.7 ± 0.7

(or 78%) are formed with the

2.7 ± 0.5 (or 22%) are established with fatty acid

tails. In summary, our ndings highlight (i) the expulsion of Aβ 25-35 from the bilayer core compared to deeper insertion of Aβ 25-35(10-40), (ii) non-uniform binding anities along Aβ 25-35 sequence, which reach maximum at the N-terminal, and (iii) important role of cationic Lys28 in binding Aβ 25-35 to the DMPC bilayer.

Aβ25

− 35

two-state binding behavior

Visual inspection of

P (z; i)

in Fig. 3a indicates that the Aβ 25-35 C-terminal occasionally

exhibits a distinct binding state, in which it becomes inserted deep into the bilayer with

z < zP .

To gain insight into potential two-state binding behavior, we display in Fig. 5 the

probability distribution

P (zcom )

of Aβ 25-35 center of mass

zcom

along the bilayer normal.

Two distinct peptide populations become apparent: the inserted state I located at 14Å and the surface-bound state S occurring at

16

zcom ≈

zcom ≈ 26Å. (Importantly, even though zcom


Page 17 of 40 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


I\

1 .0

1 .0

0.5

0.5

� ·c:-" "---

(._) Rc )

For Aβ 25-35, the bilayer is therefore


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thinned by only

∆D = 3.8 ± 1.7 Å. For comparison,

Fig. 6 also displays

nl (r, z) for Aβ 10-40

peptide bound to the DMPC bilayer revealing that the longer peptide thins the bilayer to a much greater extent. Indeed, to

12.7 ± 2.7

Å.

∆D

for Aβ 10-40 is more than three times larger being equal

50

Figure 6: The cross-sectional number densities of bilayer heavy atoms of the distance

r

to the peptide center of mass and the distance

z

nl (r, z)

The left and right sections describe the binding of Aβ 25-35 and Aβ 10-40 bilayer. Dashed lines separate proximal (r bilayer boundaries for Aβ 10-40.

zb (r)

< Rc )

and distant (r

as a function

to the bilayer midplane.

> Rc )

50

to the DMPC

bilayer regions. The

are presented by continuous black lines for Aβ 25-35 and red lines

The gure demonstrates that the binding of Aβ 25-35 induces much weaker

perturbation of the DMPC bilayer than Aβ 10-40.

To further evaluate the eect of Aβ 25-35 peptide binding on the distribution of DMPC lipids, we present in Table 2 the surface lipid number densities

ns for the distant and proximal

regions. It follows that, compared to the distant region, Aβ 25-35 binding reduces

ns

in the

proximal region by merely 13% and then by one-third in the center of binding footprint. In contrast, Aβ 10-40 causes much more profound depletion of the lipid density. According to Table 2,

ns

drops 31% in the proximal region and almost three-fold in the binding footprint

center. Relatively minor depletion of the bilayer density induced by Aβ 25-35 is consistent with modest bilayer thinning caused by this peptide and its expulsion from the bilayer core. Aβ 10-40 exhibits a dierent impact on the DMPC bilayer structure by producing a sharp

21



Page 22 of 40

drop in the lipid density, which eectively results in the formation of a void in the bilayer leaet. Table 2: Eect of Aβ peptide binding on bilayer surface lipid number density

Bilayer region Distant Proximal Center of binding footprint a

ns ,

Å

−2

Aβ 25-35

Aβ 10-40a

0.015 ± 0.000 0.013 ± 0.001 0.010 ± 0.001

0.016 ± 0.000 0.011 ± 0.002 0.006 ± 0.003

Data for Aβ 10-40 are taken from previous study 35

Lipid structure was analyzed by computing the lipid carbon-deuterium order parameter

hSCD (i)i

for each carbon

i

Fig. 7a, which compares

in the sn-2 fatty acid tails and their tilt angles

−hSCD (i)i

γ

(see Methods).

for the distant and proximal regions, reveals a minor

disordering in the proximal lipids manifested in a systematic decrease in their compared to distant values. regions,

−h∆SCD (i)i,

0.032 ± 0.003

−h∆SCD (i)i

than Aβ 10-40.

between distant and proximal

0.024 ± 0.002,

−h∆SCD (i)i

for Aβ 10-

for Aβ 10-40 is larger than the respective value

Specically, the average values of and

−hSCD (i)i

is shown in the inset to Fig. 7a, along with

40. For almost every carbon, for Aβ 25-35.

The dierence in

−hSCD (i)i

−h∆SCD i

for Aβ 10-40 and Aβ 25-35 are

conrming that Aβ 25-35 disorders the proximal lipids less

In addition, Fig.

7b compares the lipid tilt angles

γ

between the distant

and proximal regions. This gure demonstrates that Aβ 25-35 induces a minor increase in the tilt of proximal lipids compared to the distant ones. Specically, the dierence between the tilts in the two regions is

∆γ = hγiprox − hγidist = −3.6 ± 0.5◦ .

By contrast, Aβ 10-40

binding to the DMPC bilayer produces a larger increase in lipid tilt of Similar to the analysis of

−hSCD (i)i

∆γ = −4.6 ± 0.2◦ .

above, this outcome points to a greater disordering

eect induced by the Aβ 10-40 peptide. Thus, we surmise that, compared to Aβ 10-40, Aβ 2535 causes considerably smaller bilayer thinning and density depletion and, consequently, produces smaller perturbation in lipid structure.

22


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Figure 7: (a) The carbon-deuterium order parameter in

sn − 2

hSCD (i)i

computed for each carbon

i

fatty acid tails of DMPC lipids: Solid and dashed lines represent proximal and

distant lipids. The inset shows the dierence in the order parameters between proximal and

h∆SCD (i)i = hSCD (i)idist − hSCD (i)iprox , (in red). (b) Probability distributions P (γ)

distant lipids,

computed for Aβ 25-35 (in black)

and Aβ 10-40

of the tilt angles of fatty acid tails

γ

computed for the proximal (solid) and distant (dashed) lipids. Representative sampling

errors are shown by vertical bars.

Both panels imply that Aβ 25-35 binding causes minor

disordering in the lipids.

Discussion Mechanism of Aβ 25-35 binding to the DMPC bilayer The results of our REST simulations allow us to summarize the mechanism of Aβ 25-35 peptide binding to the zwitterionic DMPC bilayer. We found that upon binding the peptide helical content increases two-fold over that present in lipid-free water (namely, from

23


hHi =


0.15 ± 0.03

to

0.31 ± 0.03).

As a result, residues

i =Ile31,

Page 24 of 40

Ile32, and Gly33 in the R4 C-

terminal form stable helical conformations. The R3 N-terminal region remains less structured populated by turn and random coil conformations (hT (R3)i

0.43±0.03).

= 0.40 ± 0.03

and

hRC(R3)i =

Intrapeptide interactions are generally unstable, except for two helix supporting

contacts in R4. On average, Aβ 25-35 amino acids reside on the bilayer surface or remain unbound, but rarely penetrate the bilayer hydrophobic core. This conclusion follows from Fig. 3a, in which all amino acids

i

have their average positions

hz(i)i

occurring well above

zP ,

the boundary

between the surface and core bilayer regions. As a result almost half of Aβ 25-35 amino acids remain unbound from the bilayer. Importantly, the positions and interactions of individual amino acids with the bilayer vary considerably along the sequence. Several lines of evidence presented in the Results section have indicated that the N-terminal R3 region has stronger binding anity to the bilayer than the C-terminal R4. However, the most direct support for this conclusion comes from the analysis of Fig. 4, which conrms that a R3 residue forms, on average,

hCl (R3)i = 1.5 ± 0.1 contacts with lipid groups,

is reduced almost in half to

while in the R4 region this number

hCl (R4)i = 0.8 ± 0.1.

A remarkable feature of Aβ 25-35 binding, which is not evident in its average behavior, is a coexistence of two bound states. The probability distribution of mass

zcom

P (zcom )

of Aβ 25-35 center

along the bilayer normal in Fig. 5 unambiguously reveals two distinct states -

inserted I and surface-bound S - with divergent characteristics as illustrated in Fig. 8a. In the state I, almost the entire sequence (97%) is either surface-bound or inserted. In contrast, in the state S, only a third of the sequence interacts with the bilayer, leaving the C-terminal largely unbound and the N-terminal anchored to the bilayer.

Moreover, the peptides in

the state S are always extended (Fig. S8a), while in the state I they tend to adopt more collapsed conformations.

Finally, in the state I the peptides are tilted toward the bilayer

due to insertion of the C-terminal into the bilayer core. In contrast, in the state S Aβ 25-35 monomers tilt away from the bilayer surface. The conformational ensemble of the inserted

24


Page 25 of 40 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


state I is heterogeneous, while the state S displays less conformational diversity. In the state I, we distinguish two almost equally populated but structurally dierent substates. As Fig. 8a illustrates, the peptides in the cluster I1 are about 50% more extended than in I2 and adopt stable helical structure in the C-terminal unlike in I2. In contrast, the state S features one dominant and one minor cluster that dier with respect to helix propensity and the tilt of the C-terminal. It is instructive to evaluate the free energy landscape underpinning Aβ 25-35 bimodal binding to the DMPC bilayer. To this end, we computed the free energy prole

−RT ln(P (zcom )) expected,

G(zcom ) =

using the probability distribution of the peptide center of mass

G(zcom )

zcom .

As

in Fig. 5 demonstrates that the free energy of the surface state S is lower

than that of the inserted state I by

∆G ≈ (1.0±0.5)RT .

Importantly, the analysis of binding

energetics in Supporting Information suggests that the enhanced stability of S originates from favorable electrostatic interactions with water (Table S2). prole

G(zcom )

Furthermore, the free energy

reveals a barrier separating the I and S states. Interestingly, the free energy

barrier governing the I→S transition

∆G(I → S) = (1.8 ± 0.5)RT

smaller than the free energy barrier of the reverse transition (∆G(S

is about 1.6 times

→ I) = (3.0 ± 0.2)RT ).

Therefore, the peptide can be readily expelled from the DMPC bilayer, but its penetration into the bilayer is somewhat hindered by a larger free energy barrier. Aβ 25-35 produces an overall weak perturbation in the bilayer structure. The thinning of the bilayer is small (∆D

ns

= 3.8 ± 1.7

Å), and the decrease in lipid surface number density

under the binding footprint is minor (at most, a third in the center of proximal region).

Analysis of carbon-deuterium order parameter

hSCD (i)i

and fatty acid tail orientations also

argues that Aβ 25-35 causes relatively small disorder in the structure and tilts of proximal fatty acid tails compared to lipids unaected by the peptide. A weak impact on the bilayer structure is a consequence of the peptide's preference for binding to the bilayer surface reected in the overwhelming probability of the S state. However, this net eect obscures dramatic perturbations in the bilayer structure that do occur when the peptide adopts the

25



Page 26 of 40

inserted state I. To illustrate dierences in the bilayer disorder induced by the I and S states, we plot the respective lipid heavy atom number densities

nl (r, z)

in Fig.

8b.

The

gure exhibits a remarkable density void in the bilayer hydrophobic core occurring for the state I. In contrast, only a slight depression in the lipid density is evident for the state S. In fact, the peptide in the state S thins the bilayer by merely

∆D = 2.0 ± 0.5

Å, whereas,

strikingly, the thinning caused by the inserted peptide increases more than seven-fold to

∆D = 15.1 ± 0.4

Å. In line with these observations, the surface lipid number density

in the center of the binding footprint for the state S (ns unchanged compared to the distant region (ns

= 0.014 ± 0.000

= 0.015 ± 0.000Å−2 ).

−2

−2

) is almost

In contrast,

center of the binding footprint for the state I drops more than seven-fold to Å

Å

ns

ns

in the

ns = 0.002±0.001

. Interestingly, neither I or S states promote water incursions into the bilayer interior (see

Supporting Information and Fig. S9). Taking into account moderate free energy barriers

∆G

separating I and S states, we predict that the peptide can rapidly interconvert between the bound and inserted states. Then, this scenario suggests that the deep lipid density cavities generated by the I state may frequently icker beneath the bound

Aβ 25-35

peptide.

Impact of sequence truncation - comparing Aβ 25-35 against Aβ 10-40 Our recent studies

3436

describing equilibrium binding of the longer Aβ 10-40 monomers to the

zwitterionic DMPC bilayer served as a control for our current investigation of the truncated Aβ 25-35 peptide. As follows from Fig. 1a, Aβ 25-35 retains about half of the polar turn region (R3) and half of the hydrophobic C-terminal (R4) present in Aβ 10-40. As a consequence, Aβ 25-35 exhibits binding characteristics distinct from Aβ 10-40. We rst compare Aβ 25-35 against the entire Aβ 10-40 peptide. In the latter, with respect to lipid-free water the DMPC bilayer elevates the helix propensity more than three-fold to

hHi = 0.39 ± 0.03. 34

the lipid bilayer also increases Aβ 25-35 helicity, but only two-fold to

Binding to

hHi = 0.31±0.03.

If we

focus exclusively on the 25-35 region, then sequence truncation reduces its helix propensity in half, from

hHi = 0.59 ± 0.06

in tne bound Aβ 25-35(10-40) to

26


0.31 ± 0.03

in the bound

Page 27 of 40 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


(a) I1

I2

S2

S1

(b)

I

S

Figure 8: (a) The centroid structures of Aβ 25-35 conformational clusters observed in the inserted state I (left panels) and surface-bound state S (right panels). The S clusters dier with respect to helix propensity and the tilt of the C-terminal.

The I clusters are dis-

tinguished based on the peptide extension and stability of C-terminal helix.

Coloring of

molecules follows that of Fig. 1a. (b) Impact of two Aβ 25-35 bound states on the DMPC bilayer structure. Their impact is compared using the number density of bilayer heavy atoms

nl (r, z), distance

which is shown as a function of the distance

z

r

to the peptide center of mass and the

to the bilayer midplane. The left and right sections correspond to the inserted

I and surface-bound S states, respectively. proximal (r

< Rc )

and distant (r

> Rc )

Dashed lines indicate the boundaries between

bilayer regions. The bilayer boundaries

zb (r)

are

presented by continuous black lines. Both panels highlight minimal disturbance in the bilayer structure caused by the S state and the formation of deep lipid void resulting from the peptide penetration into the bilayer in the I state.

Aβ 25-35. Sequence truncation destabilizes and redistributes intrapeptide interactions. 40, Lys28 is involved in a stable salt-bridge with Asp23 (hC(23, 28)i

= 0.79 34 ).

In Aβ 10-

Truncation

eliminates this contact in Aβ 25-35, leaving Lys28 free to bind lipids. Consequently, Aβ 10-40

27



Page 28 of 40

Lys28 forms only 0.4 contacts, on average, with lipid groups compared to 1.6 contacts formed in Aβ 25-35. Involvement of Lys28 promotes electrostatic binding interactions. As a result, a subtle dierence between the binding energetics of Aβ 25-35 and Aβ 25-35(10-40) emerges (see Table S1): the contribution of polar and cationic amino acids is enhanced for Aβ 25-35, whereas the contribution of hydrophobic amino acids is diminished. The anity of Aβ 25-35 polar and cationic amino acids to the bilayer may be consistent with experimental studies reporting favorable binding of Aβ 25-35 to anionic lipid bilayers.

32

The most remarkable consequence of sequence truncation is related to Aβ 25-35 expulsion from the bilayer core.

Indeed, we observed very dierent binding proles between Aβ 25-

35 and Aβ 25-35(10-40): half of Aβ 25-35(10-40) amino acids are inserted and about equal fractions are surface-bound or unbound (21 and 26%).

In sharp contrast, almost half of

Aβ 25-35 amino acids remain unbound from the bilayer, whereas only the smallest fraction (25%) are inserted. Moreover, according to Table 1 in the predominant state S the number of unbound amino acids

hNu i further increases to 7.3±0.2 (or 66% of Aβ 25-35 sequence) leaving

less than one inserted. The outcome is relatively minor disruption in the bilayer structure produced by Aβ 25-35.

Specically, binding of Aβ 25-35 compared to Aβ 10-40 results in

more than three-fold smaller bilayer thinning, a 10-fold smaller lipid density depletion in the center of binding footprint, and smaller disordering and tilting of proximal fatty acid tails. In summary, sequence truncation leads to expulsion of Aβ 25-35 from the bilayer core, changes in binding energetics, considerably smaller impact on the DMPC bilayer structure, and destabilization of helical peptide states. Thus, the binding mechanisms of Aβ 25-35 and Aβ 10-40 peptides are dierent.

Comparison with previous studies The peptide fragment Aβ 25-35 is a naturally-occurring proteolytic byproduct isolated from the brains of aged patients

51

and is cytotoxic even in a monomeric, non-aggregated form.

11

Consequently, Aβ 25-35 monomer structure and interactions with lipid bilayers have been

28


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extensively probed in experimental and computational studies. Importantly, the NMR solution structures of Aβ 25-35 interacting with LiDS micelles reported.

Both NMR structures exhibit a well-ordered

52

and SDS micelles

α-helix

48

have been

spanning residues Lys28

through Leu34 and feature a disordered exible N-terminal involving Gly25 through Asn27. These experimental observations are consistent with our in silico DMPC-bound Aβ 25-35 structure presented in Fig.

2.

Indeed, the sequence interval Ile31-Gly33 featuring stable

helix in our simulations approximately corresponds to the helix region appearing in the Aβ 25-35 NMR structure.

48

Formation of helix structure has been also observed in all-atom

REMD simulations investigating Aβ 25-35 monomer in membrane-mimicking HFIP/water mixture.

53

Experiments have further shown that Aβ 25-35 can bind and insert into the lipid hydrophobic core of uncharged liposomes, and POPC/POPG membranes,

32,33,55

54

zwitterionic POPC or weakly anionic POPC/POPS

and zwitterionic DLPC multilamellar vesicles.

56

Ear-

lier experiments have suggested that Aβ 25-35 binds to the surface of mixed POPC/POPG bilayers via electrostatic interactions under the condition of low ionic strength. experiments of Dante et al

33

32

Subsequent

have detected two, almost equally populated bound states of

Aβ 25-35 peptide dened by the localization of D-Leu34 in the POPC/POPS bilayer. Specifically, in one of these states Aβ 25-35 becomes deeply embedded in the bilayer, positioning the labeled Leu34 at the distance of 6 Å from the bilayer midplane. In the second state, the peptide is apparently surface bound at the lipid-water interface. Furthermore, upon binding to zwitterionic POPC membranes the peptide is shifted toward the polar lipid headgroups placing the labeled Leu34 14 Å away from the bilayer midplane. A more recent study has described a coexistence of inserted and surface-bound Aβ 25-35 peptide populations in the weakly anionic 97:3 DMPC/DMPS bilayer.

57

Specically, it has been estimated that 40% of

the peptides become embedded in the hydrophobic region (2

≤ z ≤ 18Å),

while 60% remain

at the surface aligned parallel to the lipid bilayer. Revealing a close agreement with these experimental data, our simulations probing a pure DMPC bilayer have also identied two

29



Page 30 of 40

distinct peptide populations, inserted I (31%) and surface-bound S (69%) states. Furthermore, experiments have found that Aβ 25-35 surface-bound state has two subpopulations, which dier with respect to the exact location along the bilayer normal.

57

In agreement with

these observations, our analysis has found two conformational clusters in the S state, which dier with respect to the tilt of the C-terminal (Fig. 8a). Thus, our results appear to be in a good agreement with the experimental data discussed above.

33,57

It is also clear that

the membrane composition, including charged state and saturation level, has a signicant impact on the exact localization of Aβ 25-35 in the lipid bilayer, and our results may not be fully applicable to Aβ 25-35 interactions with unsaturated or highly anionic bilayers. Recently, several computational studies have probed Aβ 25-35 interactions with lipid bilayers. In particular, Tsai, et al.

14,17

have investigated Aβ 25-35 peptide binding to a lipid

bilayer using atomistic REMD simulations and implicit water/membrane model. They computed the two-dimensional free energy landscapes for Aβ 25-35 using as variables the distance of Aβ 25-35 center of mass to the bilayer midplane and the tilt of the C-terminal and observed several binding states. The most thermodynamically stable state occurred when the peptide binds to the bilayer hydrophilic surface with the C-terminal oriented parallel to the bilayer.

A second state with signicantly less favorable free energy was detected when a

peptide fully inserts into the hydrophobic bilayer region exhibiting a small tilt of the Cterminal (γR4

∼ 150◦ ).

Our current study is qualitatively consistent with those results. In

the predominant state S, Aβ 25-35 resides at the membrane surface featuring the average R4

◦ tilt of about 72 . The thermodynamically less stable inserted state I features the Aβ 25-35 ◦ C-terminal deeply embedded in the hydrophobic core with the tilt of about 111 . It should be noted however that the binding proles for amino acids computed in our work (Fig. 3a) and the work by Tsai et al

14

reveal dierences. Their study reported that the C-terminal as

a rule resides in the hydrophilic headgroups region, whereas the N-terminal residues are exposed to the aqueous phase. Our results in Fig. 3 suggest that Aβ 25-35 C-terminal samples the inserted state I or remains mostly unbound. These discrepancies are likely to reect dif-

30


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ferent models utilized in our studies to represent lipids and water. By using explicit solvent and all-atom representation of the DMPC lipid bilayer, our study adds atomistic details to the mechanisms of Aβ 25-35 binding to the membrane.

Conclusions Aβ 25-35 is a short fragment of Aβ peptide implicated in Alzheimer's disease, which is naturally-occurring and cytotoxic even in monomeric concentrations.

To investigate equi-

librium binding of Aβ 25-35 monomers to the zwitterionic DMPC bilayer, we have used all-atom explicit membrane and water models and REST simulations.

The impact of se-

quence truncation on the binding mechanism has been measured by utilizing as a control our previous REMD simulations, which probed binding of the longer peptide Aβ 10-40 to the same DMPC bilayer.

Our ndings can be summarized as follows.

First, binding to

the DMPC bilayer stabilizes helical structure in the peptide C-terminus, leaving the Nterminus largely disordered. Second, on an average Aβ 25-35 peptide resides on the surface of the DMPC bilayer displaying considerable variations in the binding propensities along its sequence. The N-terminus remains anchored to the bilayer, but the C-terminus adopts unbound or, occasionally, inserted states. On average, almost half of Aβ 25-35 amino acids remain unbound, whereas only a quarter penetrates the bilayer hydrophobic core. Third, an intriguing feature of Aβ 25-35 binding to the DMPC bilayer, which is obscured in average properties, is a coexistence of two bound states with strikingly dierent characteristics: a dominant surface-bound state and a less stable inserted state. In the surface-bound state, most of Aβ 25-35 amino acids become unbound and the peptide samples highly extended conformations, in which its C-terminus is typically pointed away from the bilayer. In contrast, in the inserted state the peptide with the tilted C-terminus residing in the hydrophobic core forms extensive interactions with the bilayer. Our further conformational analysis has indicated that in contrast to the surface-bound state, the inserted state is surprisingly het-

31



Page 32 of 40

erogeneous, featuring two structurally distinct, equally populated substates. Analysis of the underlying free energy landscape reveals that the inserted and surface states are separated by moderate barriers suggesting that Aβ 25-35 monomer may frequently interconvert between them. Fourth, Aβ 25-35 binding causes small thinning of the DMPC bilayer, induces minor depletion in the lipid density beneath the binding footprint, and fails to produce strong disordering in the lipid structure. These observations should be contrasted with the binding of the longer Aβ 10-40 peptide to the DMPC bilayer, whose C-terminal penetrates deep into the bilayer core, resulting in considerable thinning of the bilayer, formation of lipid density void, and disordering in lipid structure. Therefore, we conclude that the binding mechanisms of Aβ 25-35 and Aβ 10-40 peptides are dierent. We also showed that available experimental data are in good agreement with our in silico ndings.

We expect that the reported

observations concerning peptide preferred localization on the surface of the DMPC bilayer and ickering of inserted states, in which the monomer induces signicant disruption in the bilayer structure, may be used in the future to rationalize Aβ 25-35 cytotoxicity.

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.

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BINDING OF CYTOTOXIC AÎ²25-35 PEPTIDE TO THE DMPC LIPID

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