Structure of the Amyloid-� (1-42) Monomer Absorbed To Model Phospholipid Bilayers: AMolecular Dynamics Study

Charles H. Davis† and Max L. Berkowitz*,‡

Department of Biochemistry and Biophysics, UniVersity of North Carolina at Chapel Hill, Chapel Hill,North Carolina 27599, and Department of Chemistry, UniVersity of North Carolina at Chapel Hill,Chapel Hill, North Carolina 27599

ReceiVed: June 23, 2009; ReVised Manuscript ReceiVed: September 10, 2009

The amyloid-� (A�) peptide, the 39 to 43 amino acid peptide that plays a substantial role in Alzheimer’sdisease, has been shown to interact strongly with lipids both in vitro and in vivo. A�-lipid interactions havebeen proposed as a considerable factor in accelerating A� aggregation through the templating role of membranesin aggregation disorders. Previous work has shown that anionic lipids are able to significantly increase A�aggregation rate and induce a structural conversion in A� from a random coil to a �-structure that is similarto the monomer structure observed in mature fibrils. However, it is unclear if this structural change occurswith the A� monomer because of direct interactions with the lipids or if the structural change results fromprotein-protein interactions during oligomerization. We use extensive replica exchange molecular dynamicssimulations of an A� monomer bound to a homogeneous model zwitterionic or anionic lipid bilayer. Fromthese simulations, we do not observe any significant �-structure formation except for a small, unstable �-hairpinformed on the anionic dioleylphosphatidylserine bilayer. Further, we see that the Asp23-Lys28 salt bridgethat plays a role in �-hairpin formation is not substantially formed on the bilayer surface and that Lys28preferentially interacts with lipids when bound to the bilayer. These results suggest that the structural conversionseen in experiments are not due to the ordering of monomeric A� on the bilayer surface but are a result ofprotein-protein interactions enhanced by A� binding to the cell membrane.

Introduction

The amyloid-� (A�) peptide is an amyloidogenic proteinwhose aggregation has been linked to neural degeneration1-4

that is a hallmark of Alzheimer’s disease. Because of theassociation of A� with Alzheimer’s disease, A� has beenextensively studied1-4 over the past 20-30 years. In particular,the structure of the A� peptide, through its passage frommonomer to small aggregate to fibril, has been a subject of greatinterest for both experimental5-16 and computational17-31 re-searchers. Knowledge of the structure of A� at each step of theaggregation pathway will provide insight into the mechanismof aggregation that can hopefully be exploited for therapeuticbenefits. Previous research has already made significant progresson this front as the dogma of A� neurotoxicity has been shiftingaway from mature fibrils and toward small oligomers as one ofthe main neurotoxic species in Alzheimer’s disease.5,6,32 Thisknowledge places even further impact on gaining a fundamentalunderstanding of the earliest stages of A� aggregation as a majortarget for prevention of extensive neurodegeneration.

While it is clear that the first stages of A� aggregation arevital to the progress of Alzheimer’s disease, it has not yet beendetermined exactly what factors influence this initial conversionfrom monomer to oligomer. While oligomerization does occurnaturally in solution, there are other possible factors that maycatalyze this reaction. Recent work5-16 has demonstrated thatcell membranes may play a significant catalytic role in increasingA� aggregation rates. The A� peptide is derived from the

transmembrane amyloid precursor protein (APP).1-3 Uponcleavage from APP, the C-terminus1-3 of A� maintains asignificant portion of the transmembrane region of APP.Extensive experimental work5-12 has shown that A�, whenmixed with lipid vesicles of various structure and headgroupcharge, will aggregate at a much faster rate then in solution.Further results have demonstrated that this interaction betweenA� and lipids will induce a structural conversion5-16 from adisordered peptide into a peptide dominated by �-structure.Mixing A� peptide with anionic lipids, in the form of vesiclesor bilayers, has shown a substantial increase in both aggregationrate and secondary structure formation.5,9-12,14-16 Also, it hasbeen shown that extensive interactions with cell membranes canlead to substantial pore formation and disruption of ionbalance13,33 across the bilayer, which may play a role in A�neurotoxicity.

While experimental results have demonstrated an extensiveinteraction of A� with lipid bilayers, these same experimentaltechniques are limited in resolution. Circular dichroism, nuclearmagnetic resonance, electron microscopy; and other commontechniques6-16 require a low concentration to study A� withoutimmediate aggregation, which prevents these techniques frombeing able distinguish if A� is in a monomeric or smalloligmeric state. This subtle shortcoming due to the nature ofA� does cloud one aspect of these results: Is the secondarystructure change observed in experiment inherent to the A�monomer or does the structural conversion occur due topeptide-peptide interactions that are enhanced on the lipidsurface? Thus, molecular dynamics (MD) simulations wouldbe ideal for determining the structure of A� as a monomer onan atomic level.

* Corresponding author: Phone: 919-962-1218; fax: 919-962-2388;e-mail: maxb@unc.edu.

† Department of Biochemistry and Biophysics.‡ Department of Chemistry.

J. Phys. Chem. B 2009, 113, 14480–1448614480

10.1021/jp905889z CCC: $40.75 2009 American Chemical SocietyPublished on Web 10/06/2009

MD simulations have been extensively performed on the A�peptide. Simulations on the full length monomer,20,22,23,30,31 onmonomer fragments,19,24-26,28 on small oligomers17,21,24,29 andon full fibrils27 using all atom17-23,25-31 or coarse graintechniques24,29 in both explicit18,19,22,23,25-31 and implicit17,20,21,24

solvent have produced significant insight into the structure ofA� at each step of aggregation. Replica exchange MDsimulations,17-22 which allow for much more extensive motionof the peptide through its configurational space by effectivelyovercoming barriers on the conformational free energy surface,have been particularly insightful for A� structure studies. Thesereplica exchange studies17-22 on A� have shown that the peptidedoes have a predominantly random coil structure in solutionbut can still form some stable secondary structure, such as�-hairpins or small helices, that may help to accelerate aggrega-tion. However, these same studies have not yet been extendedto A� in an explicit membrane environment. Previous studieshave investigated the stability of a preinserted30,31 A� into eitheran explicit or implicit membrane environment, but these worksdid not concentrate on A� structure when bound to themembrane surface. A previous work of ours34 investigated A�binding free energies to various bilayers of differing headgroupcharge, but we did not investigate A� structure because of lackof sampling without replica exchange. In this work, we use thereplica exchange method to study A� structure and the effectof both peptide charge and lipid charge on peptide structure.As anionic lipids bilayers are able to decrease local pH,9,35 it isnecessary to study the effect of lipid binding on A� withdifferent total charges. The results of this work will help toelucidate the mechanism of structural change experimentallyobserved in A� aggregation at a molecular level not availableto many current experimental techniques.

Theoretical Methods

Initial Conditions. The initial structures used for replicaexchange simulations were obtained from a previous work34 byour group. In that work, a random coil structure of the 42 aminoacid A� peptide was systematically pulled to the surface of anequilibrated 128 lipid dipalmitoylphosphatidylcholine (DPPC)or dioleylphosphatidylserine (DOPS) bilayer using the umbrellasampling technique, and a free energy of binding was calculated.The initial conditions for the replica exchange simulationspresented here were obtained from the final snapshots of MDsimulations at the bound free energy minimum, as determinedfrom the calculated free energy profiles for binding. These finalsnapshots from the simulations occurred at an A�-bilayer center-of-mass separation of 2.1 nm with DPPC and 2.4 nm withDOPS. Thus, the initial structures chosen for the replicaexchange simulations described here represent configurationswhere the 80 ns of restrained MD simulations allowed forextensive simulation time to equilibrate the A�-bilayer system.The A�-bilayer center-of-mass restraint used in the previouswork was removed for replica exchange, ensuring that the A�was not restrained to the bilayer surface.

Peptide and Lipid Parameters. Replica exchange simula-tions were performed using the GROMACS 4.0 simulationpackage.36-39 The A� peptide was described using the united-atom GROMOS96 force field.36-38 The DPPC and DOPS lipidswere described using the Berger40 force field parameters. Theseforce fields were chosen to match our previous work34 andbecause of the extensive use of each force field in previousmolecular dynamics studies. The system was solvated withSPC/E model41 water and included counterion and co-ion Na+/Cl- salt. As the local pH near anionic lipid bilayers is lower

than bulk,9,35 it was important to perform these simulations atphysiological pH and also at a lower pH to determine if peptideprotonation state could affect A� structure near lipids. Thus,simulations were performed with A� at a -3 total charge, whichis the physiological charge of A�, and with a net 0 total chargedue to protonation of three histidine residues. This 0 total chargestate would be a close approximation of the charge on A� atpH 5, which is a feasible pH for A� bound to an anionic bilayersurface. Therefore, the -3 total charge simulations are termedthe pH 7 simulations and the 0 total charge simulations aretermed the pH 5 simulations throughout, for simplicity. TheN-terminus was represented by NH3

+, and the C-terminus wasrepresented by COO- to match the most likely charge state atphysiological pH. Thus, four total replica exchange simulationsare performed with two peptide charge states on either a DPPCor DOPS bilayer. Exact contents of the simulations are givenin the Supporting Information (Table S1). For each replica,temperature was held constant using a Nose-Hoover42 schemewith a relaxation time of 0.5 ps under constant volume (NVT)conditions. All bonds were constrained using the LINCSalgorithm,43 which allowed for a time step of 3 fs. Long rangeelectrostatic contributions were maintained using the SPMEalgorithm44 with periodic boundary conditions in all threedimensions. Secondary structure was calculated using the DSSPalgorithm45 within GROMACS and all other analysis wasperformed using GROMACS utilities. As a clarification notefor the tables and figures, a discontinuous trajectory refers to asystem trajectory obtained at a fixed temperature during thereplica exchange, while a continuous trajectory refers to atrajectory obtained by reordering the set of discontinuoustrajectories so that one initial structure is followed through thetemperature space during the full simulation time.

Replica Exchange Details. The temperature exchange simu-lations involved 83 replicas spaced between 325 K and 502 K.The exact temperatures of all replicas are provided in theSupporting Information (Table S2). The temperatures werechosen using the temperature generator for REMD-simulations46

associated with the GROMACS package. Thus, 80 ns (pH 7)or 110 ns (pH 5) molecular dynamics simulations wereperformed for each of the 83 replicas at random initial velocitieswith exchanges attempted every 3 ps and coordinates writtenevery attempted exchange. The pH 5 simulations were extendedto 110 ns to determine the stability of secondary structure formedat the end of the 80 ns simulation. With a minimum of 80 nstotal simulation time and 3 ps exchanges, over 26 000 exchangeattempts are made during the span of the simulation. Further,results from the simulations showed an exchange probabilityof 0.2-0.35 over all temperatures. The combination of a rateof exchange greater than 1 ps and over 20 000 exchange attemptsshould guarantee at least one full transit for the replicas throughthe temperature space and, subsequently, a well-mixed47 replicaexchange simulation. All details involving the replica exchangeand the replica temperatures were the same for the four initialconditions.

Bilayer Constraint. At the high temperatures used in thissimulation, neither the DPPC or DOPS bilayer was stable. Thus,a restraint was necessary to keep the fidelity of the bilayer whilestill allowing the peptide significant conformational flexibility.Further, it was important to maintain individual lipid flexibilitywithin the bilayer as lipid-protein interactions may play asignificant role in A� structure. A restraint was chosen to keepthe average separation along the Z-axis between the phosphategroups of the two leaflets of the bilayer constant. In order toimpose this restraint, the 64 phosphate atoms from each leaflet

Structure of the Amyloid-� (1-42) Monomer J. Phys. Chem. B, Vol. 113, No. 43, 2009 14481

were grouped together and a harmonic constraint was appliedto the average distance along the Z-axis between the center-of-mass of these two groups. By imposing the constraint on thecenter-of-mass separations of the group instead of each phos-phate individually, it allows for each lipid to fluctuate signifi-cantly as long as the average Z value for each leaflet remainsrelatively constant. To calculate the average constraint distancefor the DPPC and DOPS bilayers, we used the final 20 ns ofthe umbrella sampling simulations of our previous work.34 Fromthese previous simulations, we calculated that the averagephosphate-phosphate distance on DPPC was 3.90 nm and theaverage distance on DOPS was 4.16 nm. It was necessary toimpose a force constant of 4 kJ/(mol nm2) to prevent the bilayerfrom breaking apart and to prevent exceptional amounts of waterfrom penetrating the bilayer. Visual inspection of the trajectoriesat 502 K show that the bilayer remains together throughout the80 ns simulation and the extent of bilayer fluctuations are notsignificantly different in comparison to the simulation at 325K. Further, we calculated the number of water molecules thatinteract with the hydrophobic core of the bilayer. At 502 K,the number of water molecules interacting with the bilayer coreis three times larger than the number of water moleculesinteracting with the bilayer core at 325 K, and these quantitiesare stable over the length of the simulation. Both the leaflet toleaflet restraint and the constant volume (NVT) simulationcondition, which also acts as a restraint on the system, preventthe increase in water molecules near the hydrophobic core athigh temperature from disrupting the bilayer structure, thusforcing more water molecules into the hydrophobic core andeventually dissolving the bilayer. As previously mentioned, theleaflet to leaflet restraint on the bilayer was the only specificrestraint applied on the system, and the peptide was notrestrained to the bilayer surface during replica exchange.

Results

A�-Bilayer Replica Exchange MD Setup. In order toproperly assess the structure of monomeric A� at the surfaceof the bilayer, replica exchange simulations were performed forthe 42 amino acid form of the A� peptide at pH 7 (-3 totalcharge) or pH 5 (0 total charge) on either a homogeneous DPPCor DOPS bilayer. The initial structures for each of the fourreplica exchange simulations were taken as the final equilibratedstructures at the membrane-bound free energy mimima deter-mined in a previous work.34 Figure 1 is a representative exampleof one of the initial configurations for A� bound to the bilayersurface. The initial configurations for A� represent an A�structure where the peptide is strongly bound to the interfacial

region of the bilayer with some amino acid side chains insertedinto the bilayer hydrophobic core and some side chains eitherfully solvent exposed or interacting with the lipid headgroups.

MD simulations using the replica exchange methodology werethen performed using these four initial configurations over atemperature range of 325 K to 502 K. As demonstrated in Figure2, the replica exchange setup employed in this work allows eachreplica to adequately explore the temperature space during theduration of the simulation, which was consistent over all foursimulation conditions. In combination with the 3 ps exchangerate of these simulations, the extensive motion through thetemperature space should allow these replica exchange simula-tions to be considered well mixed and the local configurationalspace well sampled.47 Further analysis at the lowest temperature(325 K) should provide an appropriate estimate to the confor-mational space of A� while bound to a bilayer surface. Theuse of a weak restraint between the average positions of thephosphate groups from each leaflet of the bilayer allows forsome natural fluctuations of the bilayer surface without allowingthe bilayer to dissolve. Allowing the bilayer to be more flexiblemay play a significant role in the dynamics of a protein boundto the bilayer surface.

A� Secondary Structure on the Bilayer Surface. Secondarystructure content of the protein was calculated at 325 K for allfour systems as a means of determining how the membranesurface influences A� structure (Figure 3). From these calcula-tions, it is clear that A� remains in a predominantly randomcoil configuration throughout the duration of the simulation. Inparticular, at pH 7, the protein contains almost no orderedstructure except for a small amount of �-sheet formed near theend of the simulation of pH 7 A� on DOPS. However, at pH 5,A� is able to adopt structures containing more ordered secondarystructure. Most notably, the pH 5 protein on DOPS develops aturn/�-sheet structure from residues 21-34 beginning at 76 nsof the simulation, which can be seen more clearly in Figure4A. This structure is reminiscent of a �-hairpin, which has beenpostulated to be the building block48,49 of A� fibrils. On DPPC,it appears that a similar structure is formed, also at 75 ns, butthis structure spans fewer residues and is more transient. Thereplica exchange simulations at pH 5 on DPPC and DOPS wereboth extended 30 ns to test the hairpin stability. From Figure 3,it is obvious that this structure is still not stable over the lengthof the simulation and has quickly disappeared after 86 ns oftotal simulation time. This �-hairpin structure only appears for10 ns of the 110 ns of total simulation time and does notsignificantly influence the average structure of the peptide during

Figure 1. Initial structure of pH 7 A� peptide on DPPC bilayer. Solventand ions were removed for clarity.

Figure 2. Time series of temperature exchange for replica starting at325 K for the pH 7 A� peptide on a DPPC bilayer.

14482 J. Phys. Chem. B, Vol. 113, No. 43, 2009 Davis and Berkowitz

simulation. Finally, a turn and some �-sheet structure aretransiently formed in the last five residues of the pH 5 A� duringthe simulation on DOPS. This small turn has been previouslyseen22 in studies of A�, due to Gly37 and Gly38 which havebeen predicted to strongly favor formation of turns. These replicaexchange simulations confirm that this turn is feasible, yet it isnot stable in the interfacial membrane environment.

Asp23-Lys28 Salt Bridge Stability. The salt bridge thatcan be formed between Asp23 and Lys28 has previously beenimplicated19,25,26,48,50 to be a major factor in fibril formationby A� and has been observed in structures of A� fibrils.48 Arecent experimental work50 has demonstrated that a covalentlactam bridge created between Asp23 and Lys28 substantiallyincreases the A� aggregation rate. Further, many computationalstudies19,25,26 have shown that this salt bridge is significant inthe formation of a �-hairpin in monomeric A�. However, it isstill a point of contention if this salt bridge is necessary forfibril-like �-sheet formation in A� or if hydrophobic interactionsdrive this sheet formation and the salt bridge is a secondaryeffect of �-sheet formation. To understand the properties of thesalt bridge in our system, we calculated the distance betweenthe Cγ group of Asp23 and the N� group of Lys28 during thepH 5 A� replica exchange simulations at 325 K from 75 ns to81 ns on the DOPS bilayer, where the hairpin is transiently stable(Figure 4A). Salt bridges between these two residues havepreviously19 been described as close-contact salt bridges forseparations less than 4.5 Å and mediated salt bridges forseparations within 4.5 Å to 7 Å. The snapshots shown in Figure4B and the N�- Cγ distance plot in Figure 4C demonstrate thatA� tends to adopt one of two structures depending on theAsp23-Lys28 distance. When Asp23 and Lys28 are separatedby a significant distance (labeled (1) in the figure), A� adopts

a �-hairpin structure with a turn between residues 21-24 (Ala-Glu-Asp-Val). However, when a mediated salt bridge is formedbetween Asp23 and Lys28 (labeled (2) in the figure), the turnis shifted from residues 21-24 to approximately residues 18-22(Val-Phe-Phe-Ala-Glu) and a new, smaller turn is createdbetween Asp23 and Lys28 to accommodate this salt bridgeformation. From Figure 4C, A� appears to fluctuate betweenthese two strand-loop-strand structures over the time periodstudied and that neither structure appears to be significantly morestable then the other. Further, no close-contact salt bridgesappear to be formed between Asp23 and Lys28, in contrast tosimulations19 performed in solution that investigated this salt-bridge formation. It is important to note that these ordered�-structures were not significantly stable over the full simulationtime and were only formed with the pH 5 peptide interactingwith either bilayer.

A� Distribution on the Bilayer Surface. While the second-ary structure and salt-bridge calculations did not show asignificant difference between A� structure on the DPPC versusDOPS bilayers, we also performed density distribution calcula-tions to determine if regions of the A� peptide interacteddifferently with the zwitterionic or anionic lipids. For thisanalysis, A� was separated into a charged section (residues1-23) and a hydrophobic section (residues 24-42) to see howthe peptide interacted with the charged headgroups on the bilayersurface. In Figure 5, the left panel shows the pH 5 A� densitydistribution on DPPC. This plot shows that both the hydrophobicand charged sections of A� are interacting with the interfacialregion of the bilayer. Further, the density of the phosphate atomsin DPPC is also shown, which further supports an interfacialdistribution for the peptide. The charged and hydrophobicsections of the peptide appear to be distributed very similarly,both in the peak location and the width of the distribution. Italso appears that the hydrophobic section of the peptide is notsignificantly inserted into the bilayer core. For the distributionon the DOPS bilayer given on the right panel of Figure 5, asimilar pattern is observed. Both sections of the peptide appearto be sitting in the interfacial region of the bilayer, and thedistributions from both the charged and hydrophobic sectionsoverlap. However, one significant difference is that the hydro-phobic section of the peptide appears to have a broaderdistribution on DOPS, including significant tails on both sidesof the distribution indicating either partial insertion of thehydrophobic section into the bilayer core or more solventexposure of the hydrophobic residues at the C-terminus. Further,the charged section of the peptide also is able to becomesignificantly solvent exposed by being distributed outside of theDOPS density, which is not seen in the DPPC bilayer. Thus,while A� on the DPPC bilayer has an almost parallel arrange-ment with both sections of the peptide bound to the interfacialregion of the bilayer, A� bound to DOPS is able to adopt alarger range of distributions on the bilayer surface, including adistribution with a more exposed hydrophobic C-terminus, whichmay play a significant role in peptide-peptide interactions thatdrive aggregation.

Finally, we investigated the distribution of various aminoacids on A� with respect to the bilayer. In particular, we lookedat Lys28, which has been shown to be integral to bilayerassociation,12,16,33 as well as its importance in the Asp23-Lys28salt bridge described previously. As shown in Figure 5, Lys28tends to distribute on the interfacial region of the bilayer nearthe hydrophobic core, independent of the charge of the lipids.Interestingly, while the Lys28 density does overlap somewhatwith the phosphate density from the lipid headgroups, the lack

Figure 3. Secondary structure for each residue at 325 K for thediscontinuous simulations of (a) pH 7 A� on the DPPC bilayer, (b)pH 7 A� on DOPS bilayer, (c) pH 5 A� on the DPPC bilayer, and (d)pH 5 A� on the DOPS bilayer. The secondary structure calculationsare for the full 80 ns simulations for pH 7 A� and the full 110 ns forpH 5 A�. Secondary structure readout was produced using DSSP withinGROMACS.

Structure of the Amyloid-� (1-42) Monomer J. Phys. Chem. B, Vol. 113, No. 43, 2009 14483

of complete overlap and different density peaks for Lys28 andthe lipid phosphate atoms demonstrates that Lys28 is likely notbonding strongly to these atoms throughout the entirety of the

simulation. Further, the Lys28 distribution near the lower endof the A� hydrophobic density distribution indicates that Lys28may be playing a role in anchoring the peptide to the interfacialregion of the bilayer through interactions with the glycerolbackbone or hydrophobic tails of the lipids. These results ofthe Lys28 density distribution studies are evidence that the roleof Lys28 in A� interactions with the bilayer surface is tostabilize the interaction of A� with the interfacial region of thebilayer, thus contributing to the strong binding free energiespredicted34 for A� binding to either the DPPC or DOPS bilayersurface.

Discussion

The results of the replica exchange simulations of the A�1-42 peptide on the zwitterionic DPPC or anionic DOPS bilayersurface provides insight into the structure of the monomericpeptide when bound to a membrane. These simulations appearto be well-mixed replica exchange simulations, and the weakrestraints applied to prevent the bilayer from dissolving at hightemperature allow for significant bilayer motion, which mayinfluence peptide structure or distribution on the bilayer surface.Bilayer restraints were also used in a previous all-atom replicaexchange study51 with a peptide-bilayer system, although thoserestraints were much more constricting since the temperaturesused in the replica exchange simulations were over a muchlarger range (350 K to 800 K).

The secondary structure results from these simulations showthat, even when bound to the bilayer surface, monomeric A�

Figure 4. (a) Secondary structure for pH 5 A� on the DOPS bilayer for the discontinuous simulations at 325 K from 75 ns to 81 ns of simulationtime. (b) A� structure at time points of 77.805 ns and 78.363 ns showing the average protein structure. Residues Asp23 and Lys28 are shown inspace filling mode. (1) and (2) on the snapshots are labels for the salt bridge that could be formed between these residues. (c) Plot of the distancebetween the Cγ of Asp23 and N� of Lys28 over simulation from 75 ns to 81 ns. The labels (1) and (2) on the plot represent the Asp-Lys distanceshown in the snapshots from (b).

Figure 5. Density distribution of A� on DPPC or DOPS bilayer at325 K. The Abeta-charged density distribution represents residues 1-23of the A� peptide while the Abeta-hydrophobic density distributionrepresents residues 24-42. The phosphate density distribution representsthe distribution of phosphate atoms on the individual DPPC or DOPSlipids within the bilayer.

14484 J. Phys. Chem. B, Vol. 113, No. 43, 2009 Davis and Berkowitz

does not adopt a stable secondary structure. Many replicaexchange simulations17-22 have previously been performed usingfull-length20,22 A� or A� fragments18,19 in both explicit andimplicit solvent. These studies17-22 have shown that A� prefersa random coil structure in solution, but it is able to form stablesecondary structure, which can range from strand-loop-strandstructures to small helices, for times during the simulation. Oneof the most important aspects of these previous studies is thatA� is able to adopt a multitude of structures and is a highlyflexible peptide in solution, which is considered an importantaspect of A� aggregation, over time scales comparable to thesimulations performed in this work. However, our resultsindicate that the interactions of A� with the lipid bilayer donot promote structural ordering similar to observations madein aqueous solution and that conformational motion is likelyrestricted because of direct protein-lipid interactions. At pH7, A� does not form any stable secondary structure on eitherbilayer, while at pH 5, A� is able to form some transient�-hairpin structure, especially when bound to the DOPS bilayer.Further, our results show that different �-hairpin structures areformed when bound to DOPS, and the Asp23-Lys28 salt bridgeinteraction may play a significant role in determining whichstructure is stabilized. These results may appear contrary toexperimental results5,9-12 showing a significant increase in�-structure when A� is mixed with anionic lipid vesicles. It isimportant to note that those studies are not able to distinguishbetween A� monomers and small A� oligomers on the lipidsurface, and our results imply that the �-structure seen inexperiment is due to peptide-peptide interactions that occurduring oligomerization, which are promoted when A� is boundto the anionic lipid surface.

The results from the A� density distribution analysis alsoillustrate important aspects of A� interactions with lipid bilayers.The density distributions show that the peptide interacts stronglywith the interfacial region of the bilayer. Further, residues suchas Lys28, when not engaging in intrapeptide salt bridgeinteractions, are likely interacting with the glycerol backboneor carbon tails of lipid molecules, which stabilizes this binding.The extent of contact between the bilayer surface and A� wasalso calculated at the extreme of the replica exchange simulation,502 K (Figure S3). Even at 502 K, at least 25 of the 42 residuesof A� maintain strong contact with the bilayer surface and, onaverage, greater than 35 residues remain in contact, independentof bilayer or peptide charge. These results are not surprising inlight of our previous calculations of significant binding freeenergies for A� to the bilayer surface, which are substantiallylarger than kT even at a system temperature of 502 K. Theseresults also agree with previous molecular dynamics studies ofpreinserted A� which show either partial or full removal of atransmembrane A� from a DPPC bilayer over simulation timebecause of favorable interactions of A� with the interfacialregion30,31,34 of the bilayer. Further, previous experimental work7

has observed favorable interactions of A� with the interfacialregion of the bilayer both in full length A� and A� fragments.

The results of these replica exchange molecular dynamicssimulations provide significant insight toward the role of cellmembranes in A� aggregation. While the bilayers used in thisstudy are only simplified models of the complex lipid andcholesterol mixtures of which cellular membranes are composed,these model bilayers do provide an appropriate system for testingA�-bilayer interactions on the atomic levels available tomolecular dynamics simulations. Further, even though thereplica exchange techniques used in this work do improve uponsampling of the conformational free energy surface of A� when

bound to the bilayer, there are still limitations to the ability ofMD to overcome significant free energy barriers over the courseof a simulation. Nevertheless, in comparison to previous replicaexchange simulations17-22 of the A� peptide in solution withsimilar temperature ranges and time scales that show significantpeptide flexibility, the motion of the A� peptide is severelyretarded when bound to the bilayer surface. This comparisonbetween similar replica exchange studies of A� in solution andon the bilayer surface implies that A� interactions with thebilayer surface substantially affect A� dynamics in comparisonto A� dynamics in solution.

Further, the results of this work imply that the role of thebilayer in A� aggregation may be multifold, as has beenpreviously postulated.5,6,52 First, because of the strong bindingof A� to the bilayer surface, A� peptides will accumulate onthe surface of the bilayer. This will transiently increase localpeptide concentration and increase diffusion by limiting motionto two dimensions, which will speed up aggregation incomparison to aggregation rates in solution. Next, when boundto the anionic bilayer, the local pH will drop. As has beenobserved in vitro, A� aggregation rates increase in solution atlower pH53 and the same pattern may hold on the bilayer surface.Finally, the results imply that the hydrophobic section of A� ismore exposed and accessible on the bilayer surface, which willpromote protein-protein interactions through mutual hydro-phobic regions of adjacent A� monomers that will drive theearliest stages of aggregation. However, these results do notobserve any significant structure formation of the monomericA� peptide on the surface of the bilayer, which had beenpostulated as playing a role in the increased aggregation ratedue to lipid interactions. Our results predict that the stronglypromoted secondary structure formation in A� when mixed withlipids observed in experiment is likely due to the peptide-peptideinteractions that are greatly enhanced by interactions with thebilayer. Thus, experimental studies using highly precise methodsfor determining A� structure both in monomer and smalloligomer (dimer, trimer, tetramer) form when bound to a lipidsurface will be necessary to determine where the secondarystructure formation observed previously is originating. Further,future all-atom or coarse-grain MD studies using a similarreplica exchange setup employed in this work with multipleA� peptides bound to a bilayer in which direct peptide-peptideinteractions and potential secondary structure change are fol-lowed would also be very insightful for determining ifprotein-protein interactions are the cause of the secondarystructure change seen in experiment during A� aggregation neara membrane surface.

Conclusions

In this work, replica exchange MD simulations were per-formed on all-atom representations of the 42 amino acid A�on model lipid bilayers using novel restraints to maintain bilayerintegrity at high temperatures. The replica exchange simulationsappear to be well mixed and allow for significant conformationalfreedom for the peptide on the bilayer surface in comparison toMD simulations previously performed at 323 K.34 The resultsof these simulations show that no stable secondary structure isformed by the A� monomer at either pH 7 or pH 5 when boundto the homogeneous DPPC or DOPS bilayers. A salt bridgebetween Asp23 and Lys28, which may play a significant rolein A� aggregation, is formed on the DOPS bilayer when thepeptide has a net neutral charge, and the formation of the saltbridge imposes a �-hairpin like structure on the peptide.However, this salt bridge is not stable and this secondary

Structure of the Amyloid-� (1-42) Monomer J. Phys. Chem. B, Vol. 113, No. 43, 2009 14485

structure is not maintained because of the extensive peptide-lipidinteractions that are precluding the intrapeptide interactionsnecessary for stable secondary structure. In particular, Lys28substantially interacts with the phosphate moiety and glycerolbackbone of the lipid, which stabilizes protein binding to thelipid even at 502 K. It appears, from these results, that the stronglipid-protein interactions which force the tight binding of A�to the bilayer surface also prevent the internal interactions whichwould promote the secondary structure change observed duringA� aggregation in experiment.

The results of this work raise interesting questions regardingthe earliest stages of A� aggregation near the bilayer surface.Experimental techniques have observed significant �-structureformation when A� is incubated with anionic lipid. Ourobservations tend to indicate that this �-structure formation isnot due to structuring at a monomer level but is potentially dueto peptide-peptide interactions that are enhanced on the bilayersurface. Further, the lack of secondary structure for monomericA� bound to the bilayer surface may also shed some insightinto the large diversity of oligomers that have been observedduring A� aggregation. As the A� monomer is more extendedin its membrane-bound, unstructured form, aggregation on thebilayer surface may lead to oligomer structures that are lesscompact then oligomers observed in solution. In the least,oligomers formed on the bilayer surface should be less structuredthan oligomers in solution that are forced to have a morecompact, ordered structure due to interactions with water. Ourwork demonstrates that interactions with a membrane signifi-cantly affects structural dynamics of the A� monomer incomparison to A� in solution, which may in turn considerablyalter the pathway of A� aggregation near the cell membrane.

Acknowledgment. This work was supported by the NationalScience Foundation under grant number MCB-0615469. Theauthors thank Z. Zhang for technical assistance on moleculardynamics simulations, and the UNC Research Computing Groupfor providing and maintaining the computing resources used inthis work.

Supporting Information Available: Table S1 lists the fullcontents of all of the replica exchange simulations performed.Table S2 lists the temperatures used for replica exchange. FigureS3 provides plots of contact score over time for the discontinu-ous simulations at 502 K. This material is available free ofcharge via the Internet at http://pubs.acs.org.

References and Notes

(1) Kang, J.; Lemaire, H.-G.; Unterbeck, A.; Salbaum, J. M.; Masters,C. L.; Grzeschik, K.-H.; Multhaup, G.; Beyreuther, K.; Muller-Hill, B.Nature 1987, 325, 733.

(2) Miller, D. L.; Papayannopoulos, I. A.; Styles, J.; Bobin, S. A.; Lin,Y. Y.; Biemann, K.; Iqbal, K. Arch. Biochem. Biophys. 1993, 301, 41.

(3) Selkoe, D. J. Physiol. ReV. 2001, 81, 741.(4) Shankar, G. M. Nat. Med. 2008, 14, 837.(5) Matsuzaki, K. Biochim. Biophys. Acta 2007, 1768, 1935.(6) Aisenbery, C.; Borowik, T.; Bystrom, R.; Bokvist, M.; Lindstrom,

F.; Misiak, H.; Sani, M. A.; Grobner, G. Eur. Biophys. J. 2008, 37, 247.(7) Yip, C. M.; McLaurin, J. Biophys. J. 2001, 80, 1359.(8) Yoda, M.; Miura, T.; Takeuchi, H. Biochem. Biophys. Res. Commun.

2008, 376, 56.(9) Terzi, E.; Holzemann, G.; Seelig, J. J. Mol. Biol. 1995, 252, 633.

(10) McLaurin, J.; Chakrabartty, A. Eur. J. Biochem. 1997, 245, 355.(11) Ege, C.; Majewski, J.; Wu, G.; Kjaer, K.; Lee, K. Y. C.

ChemPhysChem. 2005, 6, 226.

(12) Bokvist, M.; Lindstrom, F.; Watts, A.; Grobner, G. J. Mol. Biol.2004, 335, 1039.

(13) Ambroggio, E. E.; Kim, D. H.; Separovic, F.; Barrow, C. J.; Barrow,C. J.; Barnham, K. J.; Bagatolli, L. A.; Fidelio, G. D. Biophys. J. 2005, 88,2706.

(14) Wong, P.; Schauerte, J. A.; Wisser, K. C.; Ding, H.; Lee, E. L.;Steel, D. G.; Gafni, A. J. Mol. Biol. 2009, 386, 81.

(15) Dante, S.; Hauss, T.; Dencher, N. A. Biophys. J. 2002, 83, 2610.(16) Chauhan, A.; Ray, I.; Chauhan, V. P. S. Neurochem. Res. 2000,

25, 423.(17) Anand, P.; Nandel, F. S.; Hansmann, U. H. E. J. Chem. Phys. 2008,

128, 165102.(18) Wu, C.; Murray, M. M.; Bernstein, S. L.; Condron, M. M.; Bitan,

G.; Shea, J. E.; Bowers, M. T. J. Mol. Biol. 2009, 387, 492.(19) Baumketner, A.; Shea, J. E. J. Mol. Biol. 2007, 366, 275.(20) Yang, M.; Teplow, D. B. J. Mol. Biol. 2008, 384, 450.(21) Jang, S.; Shin, S. J. Phys. Chem. B 2008, 112, 3479.(22) Sgourakis, N. K.; Yan, Y.; McCallum, S. A.; Wang, C.; Garcia,

A. E. J. Mol. Biol. 2007, 368, 1448.(23) Tomaselli, S.; Esposito, V.; Vangone, P.; van Nuland, N. A. J.;

Bonvin, A. M. J. J.; Guerrini, R.; Tancredi, T.; Temussi, P. A.; Picone, D.ChemBioChem 2006, 7, 257.

(24) Dong, X.; Chen, W.; Mousseau, N.; Derreumaux, P. J. Chem. Phys.2008, 128, 125108.

(25) Reddy, G.; Straub, J. E.; Thirumalai, D. J. Phys. Chem. B 2009,113, 1162.

(26) Tarus, B.; Straub, J. E.; Thirumalai, D. J. Am. Chem. Soc. 2006,128, 16159.

(27) Buchete, N.-V.; Tycko, R.; Hummer, G. J. Mol. Biol. 2005, 353,804.

(28) Cruz, L.; Urbanc, B.; Borreguero, J. M.; Lazo, N. D.; Teplow, D. B.;Stanley, H. E. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18258.

(29) Urbanc, B.; Cruz, L.; Ding, F.; Sammond, D.; Khare, S.; Buldyrev,S. V.; Stanley, H. E.; Dokholyan, N. V. Biophys. J. 2004, 87, 2310.

(30) Xu, Y.; Shen, J. J.; Luo, X. M.; Zhu, W. L.; Chen, K. X.; Ma,J. P.; Jiang, H. L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5403.

(31) Lemkul, J. A.; Bevan, D. R. Arch. Biochem. Biophys. 2008, 470,54.

(32) Klein, W. L.; Stine, W. B.; Teplow, D. B. Neurobiol. Aging 2004,25, 569.

(33) Arispe, N.; Diaz, J. C.; Simakova, O. Bioch. Biophys. Acta 2007,1768, 1952.

(34) Davis, C. H.; Berkowitz, M. L. Biophys. J. 2009, 96, 785.(35) Van Klompenburg, W.; de Kruijff, B. J. Membr. Biol. 1998, 162,

1.(36) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem.

Theory. Comput. 2008, 4, 435.(37) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.;

Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701.(38) Lindahl, E.; Hess, B.; van der Spoel, D. Comput. Phys. Commun.

1995, 91, 43.(39) Hukushima, K.; Nemoto, K. J. Phys. Soc. Jpn. 1996, 65, 1604.(40) Berger, O.; Edholm, O.; Jahnig, F. Biophys. J. 1997, 72, 2002.(41) Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. J. Phys. Chem.

1987, 91, 6269.(42) Nose, S. J. J. Chem. Phys. 1984, 81, 511.(43) Hess, B. J. Chem. Theory Comput. 2008, 4, 116.(44) Essman, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;

Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577.(45) Kabsch, W.; Sander, C. Biopolymers 1983, 22, 2577.(46) Patriksson, A.; van der Spoel, D. Phys. Chem. Chem. Phys. 2008,

10, 2073.(47) Abraham, M. J.; Gready, J. E. J. Chem. Theory Comput. 2008, 4,

1119.(48) Petkova, A. T.; Ishii, Y.; Balbach, J. J.; Antzutkin, O. N.; Leapman,

R. D.; Delaglio, F.; Tycko, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16742.(49) Petkova, A. T.; Yau, W. M.; Tycko, R. Biochemistry 2006, 45,

498.(50) Sciarretta, K. L.; Gordon, D. J.; Petkova, A. T.; Tycko, R.; Meredith,

S. C. Biochemistry 2005, 44, 6003.(51) Nymeyer, H.; Woolf, T. B.; Garcia, A. E. Proteins: Struct. Funct.

Bioinf. 2005, 59, 783.(52) Gorbenko, G. P.; Kinnunen, P. K. J. Chem. Phys. Lipids. 2006,

141, 72.(53) Kirkitadze, M. D.; Condron, M. M.; Teplow, D. B. J. Mol. Biol.

2001, 312, 1103.

JP905889Z

14486 J. Phys. Chem. B, Vol. 113, No. 43, 2009 Davis and Berkowitz