[Molecular Medicine and Medicinal Chemistry] Alzheimer's Disease: Insights into Low Molecular Weight...

November 26, 2012 10:1 9in x 6in Alzheimer’s Disease: Insights Into Low Molecular … b1377-chB4

4Exploring the Structuresof β-Amyloid Oligomers

in Aqueous Solution UsingCoarse-Grained Protein Models

Yassmine Chebaro∗ and Philippe Derreumaux∗,†

4.1 Introduction

Identifying the structures of the oligomers of the β-amyloid peptideis of utmost importance for efficient drug design. We report recentcomputational studies aimed at characterizing the free energy landscapes ofAβ oligomers in aqueous solution. These computer simulations are madepossible by the use of simplified protein models and efficient samplingmethods.

Alzheimer’s disease (AD) is characterized pathologically by abnormallyhigh levels of brain lesions (senile plaques made of the 39–43 amino acidβ-amyloid (Aβ) protein) and neurofibrillary tangles inside neurons madeof the tau protein. Two main directions for the development of AD drugs are

∗Laboratoire de Biochimie Théorique, UPR9080 CNRS, IBPC, 13 rue Pierre et Marie Curie,75005, Paris, France.†Institut Universitaire de France 103 Blvd. Saint-Michel, 75005, Paris. France. Tel: 33 1 5841 51 72, FAX: 33 1 58 41 50 26; E-mail: [email protected].

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284 Y. Chebaro and P. Derreumaux

pursued by researchers and pharmaceutical companies. The first involvestherapeutics targeted towards tau, as hyperphosphorylation of tau is linkedto the onset of AD. All clinical phase III studies on drugs targeting tau havefailed thus far to improve the mental state of patients. The second directionwithin the amyloid hypothesis attempts notably to modulate/block theproduction of Aβ from the large transmembrane amyloid precursor proteinby β-secretase or γ-secretase inhibitors, or inhibit Aβ aggregation and theproduction of neurotoxic assemblies.

Despite the large number of γ-secretase and β-secretase inhibitors,anti-Aβ42 vaccines, and compounds reported to interfere with Aβ aggre-gation and toxicity (De Strooper et al., 2010; Aguzzi and O’Connor, 2010),no treatment has been shown to slow the progression of AD. Clearly allof these negative results pose questions related to the amyloid cascadetheory that holds that the generation of Aβ triggers tau fibrillary pathology(Abbott, 2008). However, the hypothesis for AD causation for which thegreatest clinical and experimental support exists is that Aβ oligomers arethe primary neurotoxic species (Walsh and Selkoe, 2007). Although thesmallest agent is a dimer and toxicity increases from dimer to tetramerin cultured cells (Ono et al., 2009), neurotoxicity isolated from braintissues also originates from higher molecular weight aggregates, such as thedodecamer (Lesné et al., 2006), and fibril fragmentation also contributes toamyloid toxicity in cells (Xue et al., 2009).

The Aβ42 oligomers are known to be more toxic than Aβ40 oligomersand impair memory by disrupting memory-related functions of synapticjunctions between neurons. The ratio of Aβ40:Aβ42 differs by ten-foldbetween brains from non-demented controls and those with sporadicAD. The neurotoxicity of Aβ peptides is also induced by small changesin the Aβ42:Aβ40 ratio (Kuperstein et al., 2010). Aβ binds to andinfluences the function of many proteins such as the receptor for advancedglycation endproducts and the Frizzled receptor, among others (Sakonoand Zako, 2010). Recently, it has been shown that Aβ oligomers inducethe abnormal accumulation and over-stabilization of the metabotropicglutamate receptor 5 (Renner et al., 2010). It has also been suggested thatthe cellular prion protein (PrPC) is a mediator of Aβ oligomer-inducedsynaptic dysfunction (Laurén et al., 2009). Ablation or over-expression ofPrPC has, however, no effect on the impairment of hippocampal synaptic

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Exploring the Structures of β-Amyloid Oligomers in Aqueous Solution 285

plasticity in a transgenic model of AD, challenging the role of PrPC as amediator of Aβ toxicity (Callela et al., 2010).

Irrespective of the molecules mediating the Aβ oligomer adverse effects,if effective drug design strategies targeting Aβ are to be developed, highatomic resolution of the structures of the oligomers must be obtained. Thisis, however, a very difficult task by experimental and theoretical means.

4.2 Experimental Study of β-Amyloid Oligomersand Final Products

The Aβ42 peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL-MVGGVVIA) contains two hydrophobic patches, Leu17–Ala21 (or centralhydrophobic core (CHC)) and Ala30–Ala42, separated by a hydrophilicpatch, Glu22–Gly30. There are currently seven familial AD variantsreported: English (H6R), Tottori (D7N), Flemish (A21G), Arctic (E22G),Dutch (E22Q), Italian (E22K), and Iowa (D23N).

The kinetics of wild-type Aβ aggregation in vitro are described by anucleation–polymerization process, i.e. a lag phase of several days withoutany thioflavin binding signal until the formation of a nucleus from whichfibril growth is rapid (Harper and Lansbury, 1997). This implies that theearly Aβ oligomers are difficult to characterize at the atom level usingexperiments, because they are metastable and transient.

Aβ40 and Aβ42 are very difficult peptides and different preparationmethods can produce different conformer populations. Using filtrationwith a 10,000 molecular weight cut-off, circular dichroism (CD) of all lowmolecular weight Aβ1-40 aggregates gives 88% of random coil and β-turnand thus only 12% of β-strand at 295 K, pH 7.5, and day 0, emphasizing theheterogeneity of the energy landscape (Kirkitadze et al., 2001). In contrast,different preparations give higher β-strand content, varying from 25% formonomers to 45% for tetramers (Ono et al., 2009).

The populations of the oligomers also vary from Aβ40 to Aβ42and with the experiments used (Bernstein et al., 2009). In addition,the rate-limiting step, associated with the formation of a nucleus, varieswith protein concentration, salt, metal ions, agitation, pH, temperature,cholesterol, lipids, and chemical or amino acid modifications, indicatingdifferent assembly pathways leading to the nucleus. Of particular interest

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is the observation that both a lactam bridge connecting Asp23 and Lys28(Sciarretta et al., 2005), and the D23N substitution (Tycko et al., 2009)suppress the lag phase of Aβ1-40 polymerization.

As a result, we have little information on the structures of Aβ oligomersin real solution conditions where the aggregation is rapid. The currentchallenge is to stabilize homogeneous oligomers and glean their populationsand structures. Various conditions have been described including lowtemperature and low salt (Ahmed et al., 2010), sodium dodecylsulfate(Yu et al., 2009), and in situ chemical cross-linking to prevent oligomerdissociation or growth (Ono et al., 2009). For instance, using smallamounts of aliphatic hydrocarbon chains of detergents or fatty acids,soluble N-Met–Aβ1-42 preglobulomer (∼4 peptides/soluble aggregate)and globulomer (∼12-16 peptides/soluble aggregate), which alter synapticactivity, could be stabilized (Yu et al., 2009). The three-dimensional nuclearmagnetic resonance (NMR) spectra of the preglobulomer point to a mixedparallel and antiparallel β-sheet structure that is different from fibrils. Theprotection factors of the globulomer by amide exchange experiments arealso consistent with this description. Figure 4.1 shows the conformationsof residues 17-42 in the Aβ42 fibril model (Fig. 4.1a) and preglobulomer(Fig. 4.1b), residues 1-16 being disordered in both systems. The orientationof the two β-sheet segments within the preglobulomer is not well definedby the NMR data. Each structure displays an interstrand parallel β-sheetin the C-terminal 34-42 region. In contrast to the fibril, the preglobulomerhas an intrastrand antiparallel β-sheet connected by a loop between Val24and Asn27. A recent investigation using solid-state NMR together withsystematic proline replacement, however, differentiated the toxic conformerwith a turn at positions 22 and 23 in Aβ42 aggregates from the non-toxic one with a turn at positions 25 and 26; the former showed potentaggregative ability and neurotoxicity (Masuda et al., 2009). Overall, itremains to be determined whether all of these oligomers are relevant toin vivo amyloidosis, but they offer possible structural targets for drugdesign.

Finally, Aβ amyloid fibril insolubility makes high-resolution structuredetermination impossible and the current models based on solid-stateNMR, hydrogen-deuterium (H/D) exchange and mutagenesis, and electronmicroscopy are obtained using a limited number of observables. All of

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Figure 4.1. Conformational diversity of the β-amyloid (Aβ) peptide. (a) Two strand–loop–strand motifs (residues 17-42 are shown) from the model structure of the Aβ1-42 fibril(Lührs et al., 2005). (b) Conformations of the N-Met–Aβ1-42 preglobulomer (residues 15-42 are shown) formed in sodium dodecyl-sulphate (SDS)-containing solvent (Yu et al.,2009). (c and d) The first two dominant clusters of the Aβ17-42 trimer predicted byreplica exchange molecular dynamics–OPEP (Chebaro et al., 2012). Yellow balls show thepositions of Leu17 in the peptides. In every model, the chains are distinguished by theircolors.

the models for purely synthetic fibrils of Aβ1-40 and Aβ1-42 display across-β-structure with parallel β-sheets and β-strand–loop–β-strand (SLS)motifs, but with significant differences. Residues 1-8 are disordered in theAβ1-40 model (Petkova et al., 2002) vs. residues 1-15 in the Aβ1-42 model(Lührs et al., 2005). The β-strands S1 and S2 cover residues 12-24 and 30-40in Aβ1-40 vs. 16-24 and 31-42 in Aβ1-42. The salt bridge between Asp23and Lys28 and the side-chain interactions are intramolecular in Aβ1–40but intermolecular in Aβ1-42, and the inter-β-sheet side-chain interactionsalso vary. Interestingly, seeded growth of Aβ fibrils from AD brain-derivedfibrils produces a distinct fibril structure (Paravastu et al., 2009) and purelysynthetic fibrils of Aβ1-40 D23N have an antiparallel β-sheet structure(Tycko et al., 2009). Taken together, the data are strong evidence that themorphology, β-sheet arrangement, and detailed structures of fibrils areencoded in diverse Aβ oligomers and may originate from kinetic control(Pellarin et al., 2010).

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4.3 Computer Simulations for Amyloid Protein Aggregation

Atomistic molecular dynamics (MD) simulations offer the most detailedenergetic and dynamic picture of proteins and the surrounding water. Usinga specially built supercomputer (Shaw et al., 2010) it is possible to followthe folding process of a single protein of 60 amino acids within 0.1–1 ms.This computer resource is still not sufficient, however, for Aβ oligomers andall-atom MD in explicit solvent is used to study the stability of preformedassemblies for 100 ns (Ma and Nussinov, 2002; Masman et al., 2009; Wuet al., 2010; Miller et al., 2010a) and the early aggregation steps of smallpeptides (Nguyen et al., 2007). To push the boundaries in timescales andsizes of the aggregates, it is essential to go beyond MD and the all-atomdescription.

One of the more frequently used approaches to overcome the multipleminima problem is replica exchange molecular dynamics (REMD), whereN copies or replicas of the system are simulated by MD in parallel, eachat its own temperature, and exchanged at regular time intervals using theMetropolis criterion (Sugita and Okamoto, 1999). Because REMD allowsconformations to move between various temperatures, a better descriptionof the thermodynamics is reached at the cost of the loss of dynamics.In practice, the scaling of the number of replicas with the square rootof the total number of degrees of freedom and the ruggedness of theenergy surface lead to converged properties only for short all-atom peptidesin explicit solvent (De Simone and Derreumaux, 2010). Other generictechniques include the so-called Hamiltonian temperature REMD wherethe additional variable is an external force, discrete MD (DMD) as describedbelow, and Monte Carlo simulation where a run is a series of random stepsin conformation space, each perturbing some degrees of freedom of themolecule, and the newly generated state at each step is accepted using theMetropolis condition. The activation–relaxation technique (ART), whichmoves the system through well-defined transition states, has also been used(Mousseau and Derreumaux, 2005).

One solution for reducing the number of degrees of freedom isprovided by continuum solvent models (Mitternacht et al., 2010) such asthe generalized Born (GB) with or without solvent-accessible surface area(SA) while retaining the full atomic description of the peptides (Khandogin

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and Brooks, 2007; Anand et al., 2008; Jang and Shin, 2008; Yang and Teplow,2008). Alternatively, it is possible to employ coarse-grained (CG) models,which make use of beads to represent groups of atoms, and optimize theenergy function and parameters to include solvent effects implicitly.

The first CG model is the four-bead protein model including thebackbone N, the Cα, and the carbonyl groups with a side-chain bead(Cβ) coupled to DMD where all interparticle interactions are expressed bysquare-well and step-like potentials (Urbanc et al., 2004, 2010). Backbonehydrogen bond interactions are implemented, and side-chain–side-chaininteractions are derived from the hydropathy scale of Kyte and Doolitle(1982). In DMD, a collision occurs when two particles reach a distanceat which the potential is discontinuous. The pair of particles with theshortest collision time is chosen as the next collision event and the newpositions and velocities of the two particles involved are calculated basedon conservation laws for the linear momentum, angular momentum, andtotal energy. DMD therefore avoids the integration of Newton’s equation.Although the relationship between the DMD simulation and real time maynot be a simple linear function, current DMD simulations correspond to1–5 ms (Urbanc et al., 2010; Yun et al., 2010).

The second CG model is the six-bead optimized potential for efficientpeptide structure prediction (OPEP) model (Maupetit et al., 2007)including the backbone N, Cα, C, O, and H atoms with one side-chain beadcoupled to ART (Santini et al., 2004), MD (Derreumaux and Mousseau,2007), and REMD (Melquiond et al., 2008; Chebaro et al., 2009a) whereall particle interactions are modeled by knowledge-based potentials. Incontrast to the DMD force field, the OPEP effective potential has beenshown to mimic the structures and thermodynamics of many non-amyloidsystems (Chebaro et al., 2009a; Maupetit et al., 2009). Whether DMDand OPEP simulations reflect all of the dominant energetic aspects of Aβ

oligomers in test tubes remains to be further explored in the light of the roleof discrete water molecules in mediating oligomers and fibril formation asrevealed by a recent experimental study (Straub and Thirumalai, 2010).

Finally, it is possible to use a coarser and more extreme description,where the peptide has a single degree of freedom with two energyminima corresponding to amyloid-competent and amyloid-protectedstates (Pellarin et al., 2007). Although this model offers the possibility of

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generating amyloid fibril topologies resembling those observed experimen-tally (e.g. the twist along the fibril axis and the multifilament composition),it lacks sequence and atomistic detail.

4.4 Free Energy Landscapes of β-Amyloid Oligomers

Here, we report the results of extensive all-atom explicit solvent and CGsimulations aimed at determining the structures and free energy surfacesof Aβ oligomers in aqueous solution. We limit this to Aβ1-40/1-42 and thepeptides containing at least the amino acids 16-35.

4.4.1 β-Amyloid monomer

The Aβ1-40 and Aβ1-42 monomers are found to be highly disorderedby NMR with a weak β-strand signal at positions 17-21 and 31-36, andweak turn/bend structures at positions 7-11 and 20-26 (Hou et al., 2004).Limited proteolysis coupled to mass spectrometry suggests that residues 21-30 are protease-resistant in Aβ1-42. CD spectroscopy indicates that bothalloforms have distinct conformations at the C-terminus.

Using all-atom REMD simulation with the GB implicit model and16 replicas between 276 and 400 K for 110 ns, Yang and Teplow (2008)found that neither monomer is unstructured at 298 K, but rather behavesas a unique statistical coil with five relatively independent folding unitscomprising residues 1-5, 10-13, 17-22, 28-37, and 39-42, which areconnected by four turn structures. The two populated turns, predictedat positions 6-9 and 23-27, are in agreement with NMR. The conformationof residues 21-30 is very similar to that obtained for the Aβ21-30 andAβ16-35 monomers by NMR and REMD simulations (Baumketner andShea, 2007), respectively. The free energy surfaces show two large basinswith many minima displaying either substantial α-helix or β-sheet content.The two residues Ile41 and Ala42 increase contacts within the C-terminusand between the CHC core and the C-terminus leading to a more structuredC-terminus.

This enhanced rigidity and β-sheet content of the C-terminus inAβ1-42 with respect to Aβ1-40 are consistent with all-atom REMD simu-lations in explicit solvent using various force fields (Sgourakis et al., 2007),and are also supported by DMD simulations, with Aβ1-42 displaying a turn

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centered at Gly37–Gly38 and a β-hairpin at Val36–Ala42 that are absent inAβ1-40 (Lam et al., 2008). DMD simulations capture two other differencesbetween the alloforms: a highly populated β-strand at Ala2–Phe4 in Aβ1-40but not in Aβ1-42, and a β-hairpin centered at Ser8–Tyr10 in Aβ1-42but not in Aβ1-40 (Lam et al., 2008). A recent extensive all-atom REMDsimulation of Aβ1-42 in water (225 ns per replica) reports, however, thefrequent occurrence of β-sheets involving interactions of the C-terminuswith other parts of the sequence (Sgourakis et al., 2011).

Using all-atom Monte Carlo simulations and a home-made implicitsolvent, a fully different conformational picture was proposed. Aβ1-42preferentially populates two major topologies, either elongated withhigh β-sheet content and forming a four-stranded antiparallel β-sheetor compact with lower β-sheet content and forming two layers withmixed parallel/antiparallel arrangements (Mitternacht et al., 2010). Aβ1-42structures display three major turns at positions 13-16, 23-26, and 35-38.The first turn enables a well-defined β-hairpin spanning the residues 4-20.In contrast, the turn 23-26 leads to predominantly β-hairpin and to alesser extent coil conformations in the region 17-32, whereas the 35-38turn enables the formation of multiple β-hairpins in the C-terminus.

Based on ART–OPEP simulations, Aβ1-42 was found to populate fourdistinct conformations (Melquiond et al., 2008). One is fully random coiland the other three are mostly random coil (70% of the residues) and displayβ-strands at various positions: 3-4, 12-13, 16-19, and 25-28; or 8-12, 17-21,and 29-32; or 3-6, 12-16, 31-32, and 39-40. The CHC does not show a highpropensity for β-sheet (∼25%), in agreement with NMR spectroscopy.There is also a non-negligible signature of a turn at Gly37–Gly38 and saltbridges between Asp22 (Glu23) and Lys28.

Overall, each of these simulations reports that Aβ1-40 and Aβ1-42monomers are described by a distinct ensemble of predominantly randomcoil structures in dynamic equilibrium, consistent with NMR and CDstudies. They reach, however, divergent conclusions on the nature ofthe different conformations that characterize the whole ensemble andin particular the formation probability of structural elements resemblingthose in the fibrils: the SLS motif and the salt bridges stabilizing the loop(Melquiond et al., 2008; Straub and Thirumalai, 2010; Vitalis and Caflisch,2010; Sgourakis et al., 2011).

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4.4.2 β-Amyloid dimer

Dimers are the first toxic species disrupting cognitive function. Conver-gence of all-atom Aβ1-40/42 dimer simulations in explicit solvent beingout of reach with current computer resources, all determined free energylandscapes for full-length Aβ or shorter congeners have resorted to CG orall-atom models with implicit solvent.

It is not clear to what extent the results on Aβ10-35, Aβ9-42,and Aβ16-35 can be extrapolated to full-length Aβ. On the one hand,the deletion of residues 1-9 was shown to be marginal in terms ofstructures and energetics at 360 K from all-atom REMD simulationsusing the CHARMM19 force field, an implicit solvent, and 24 replicasfrom 300 to 530 K, each of 0.8 µs (Takeda and Klimov, 2009). On theother hand, the H6R and D7N variants produce oligomers that displayedsubstantial β-strand (H6R) or α/β (D7N) structure, and alter Aβ1-40/42assembly at its earliest stages (Ono et al., 2010). The deletion of theC-terminal hydrophobic region 36-42 must also be taken with caution,the residues Ile41 and Ala42 impacting the aggregation kinetics. Despitethese uncertainties, we believe that the peptides Aβ10-35, Aβ9-42, andAβ16-35 enable the addressing of fundamental questions pertaining to thepopulation of the aggregation-prone N∗ structures.

Using REMD with the all-atom AMBER96 force field and GBSA and32 replicas within 280 and 405 K, each of 146 ns, various Aβ10-35 dimertopologies with an averaged β-sheet content of 40% were detected at 300 K(Jang and Shin, 2006). The dominant structure with a population of 31% isa planar, each Aβ10-35 unit forming two β-strands joined by a turn region.The assembly of such bend double β-strands exhibits several differentinterlocking patterns. It is known, however, that the AMBER96 force fieldover-estimates the β-strand propensity.

Using all-atom implicit solvent REMD and 16 replicas of 90 ns inthe range 250–500 K, it was found that the Aβ1-39 dimer at 300 K iswell described by a dominant configuration that has parallel N-terminals,high α-helical content, and a well-defined segment Leu17–Ala21 that arestabilized by salt bridges between Lys28 of one chain and either Glu22or Asp23 of the other chain (Anand et al., 2008). Formation of thesesalt bridges was suggested to be the rate-limiting step in polymerization.

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However, the AMBER99 force field is known to over-stabilize α-helices andthe simulation may not have fully converged within 90 ns.

The impact of the Flemish mutation A21G on Aβ9-40 and Aβ9-42dimers was investigated by temperature-mediated unfolding MD using theall-atom GROMOS96 force field at neutral pH (Huet and Derreumaux,2006). These simulations, starting from the fibril model with theintramolecular Asp23–Lys28 salt bridge formed, show that all of theintramolecular and intermolecular salt bridges between residues Glu22(or Asp23) and Lys28 are populated to some extent in dimers, but higheroligomers are necessary to stabilize the salt bridges in their amyloid fibrilconformations.

The Aβ1-42 dimer was explored by REMD–OPEP starting from thefibril structure with the residues 1-9 extended. Using 32 replicas between290 and 650 K, each for 130 ns and excluding the first 30 ns, three resultsemerge at 300 K (Melquiond et al., 2008). First, the conformationalensemble shows β-sheet and α-helical contents of 20% and 5% consistentwith experiments (Bitan et al., 2003). Second, the probability of formingβ-strands in the CHC and 30-42 regions is very small, although there areintramolecular interactions between CHC and (Met35, Val40, and Ile41)and intermolecular side-chain interactions between the CHC regions. As aresult, the Aβ1-42 dimer does not encode the SLS motif. Finally, the residuesGlu3–His6 and Tyr10–Val12 have a probability of 30% of forming a β-sheetin parallel register. The prediction that the N-terminal region is involved inthe early Aβ aggregation steps is supported by recent experiments showingthat D7N and H6R mutations impact the secondary structure of Aβ1-42oligomers (Ono et al., 2010).

Following an earlier study (Urbanc et al., 2004), Aβ1-40 and Aβ1-42self-assembly was reinvestigated by DMD starting from 32 unfoldedpeptides using the four-bead protein model and various ratios between thestrengths of the hydropathy and hydrogen-bonding interactions (Urbancet al., 2010). The simulation gives an oligomer population-averaged β-sheetcontent of 17–20% for both alloforms and their E22G variants consistentwith experiments, and for dimers an averaged value varying between 16%(Aβ1-42) and 22% (Aβ1-40 E22G). Examining the β-strand propensityper residue, Aβ1-40 and Aβ1-42 dimers differ at the N-terminal region

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Ala2–Phe4, where Aβ1-40 but not Aβ1-42 shows β-strand propensity ofup to 0.3. Aβ1-42 dimers show a higher β-strand signal at Arg5–His6,Gly9–Glu11, Leu17–Ala21, and Val39–Val40, and a turn structure centeredat Gly37–Gly38 that is absent in Aβ1-40 dimers.

A lactam bridge between the side-chains of Asp23 and Lys28 increasesthe Aβ1-40 fibrillogenesis rate by three orders of magnitude. This ledto the hypothesis that the formation of a bent structure within residues23-29 could be the rate-limiting step in Aβ kinetics (Sciarretta et al.,2005). Although all-atom simulations of Aβ1-40 and Aβ1-35 monomers(Baumketner and Shea, 2007; Yang and Teplow, 2008) suggest that theloop 21–30 could appear early in the aggregation process, quantitativeinformation regarding its presence in small oligomers remains elusive. Tworecent simulations looked at the population of the fibril-competent loopand SLS motif in dimers.

All-atom explicit solvent MD simulations of the Aβ10-35 dimer,monomer and dimer of Aβ10-35 with constrained Asp23–Lys28 salt bridgehave been used to explore the origin of the observed enhanced rate offibril formation (Reddy et al., 2009). The simulations of 60-100 ns usingthe CHARMM22 and OPLS force fields show that the fibril-competentmonomers, N∗, are populated to a greater extent in Aβ10-35 with a lactambridge than in wild-type Aβ10-35, which has negligible probability offorming N∗, and the salt bridge in N∗ is hydrated. By revealing a reduction inthe free energy barrier to fibril formation, resulting from a decrease in theentropy of the unfolded state and the lesser penalty for conformationalrearrangement, those simulations clearly explain why the rate of fibrilformation is so much enhanced in the lactam bridge congener.

Coarse-grained OPEP–REMD has been used to explore the populationof the N∗ state in the monomer and dimer of Aβ16-35 (Chebaro et al.,2009b). At low temperature, the monomer is dominantly turn and coil,and has a negligible probability of forming a β-hairpin matching theconfiguration adopted by residues 16-35 of Aβ1-40 interacting withthe affibody ZAb3. The loop 22-28 is similar to that observed in theNMR structure of Aβ21-30 NMR and all-atom simulations of Aβ1-40/42monomers (Yang and Teplow, 2008). Interpeptide interactions impactthe global structure, but have little effect on the region 22-28, providingstrong evidence that the region 22-28 acts as a quasi-independent loop.

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The dimer populates equally the antiparallel and parallel orientations ofthe N-terminal regions 16-21 with little β-sheet structure, and leaves theC-terminal regions 31-35 unstructured and in parallel orientation. Thisresult suggests that the SLS is not populated in Aβ16-35 dimers.

4.4.3 β-Amyloid oligomers: from trimers to hexamers

Owing to the very large number of degrees of freedom, a few simulationshave explored the structures of trimers to hexamers for Aβ1-40/42 orrelevant fragments.

Based on the AMBER/GBSA force field and 32 replicas between 283and 405 K, each of 150 ns for the trimer and 112 ns for the tetramer,multiple Aβ10-35 topologies were found (Jang and Shin,2008). The startingconformation of each chain was the NMR structure of Aβ10-35 with five β-turns and one γ-turn. The β-sheet content at 300 K (50%) is much higherthan the CD value of 12% at 295 K (Kirkitadze et al., 2001). Using thelast 20 ns of the trajectories, tetramers and dimers are the most populatedspecies at 283 and 300 K, respectively. At 283 K, tetramers can be describedby two states representing 50% of the conformations, and notably anordered β-sheet displaying four SLS motifs. It would be profitable to repeatthese simulations with the OPLS force field known to better balance α/βpropensities.

Two recent all-atom MD simulations in explicit solvent were used toexplore the stability of various aggregates starting from the fibril state. First,the structures and dynamics of trimers and pentamers of Aβ1-42 with theresidues 1-17 initially extended were studied using five MD runs, each for100 ns with the OPLS–SPC force field (Masman et al., 2009). It is found thatoligomers are stable at 310 K in their fibril state and the Asp23–Lys28 saltbridges (solvated by narrow water channels) and SLS motif are maintained.Those MD also reveal that the S2 β-strand (residues 31-42) forms a tightlyorganized β-helix, whereas the S1 strand (residues 18-26) is much moremobile, maintaining its initial conformation less than 30% of the time,leading to the hypothesis that the formation of C-terminal β-sheets is acrucial event in the aggregation.

Second, all-atom 100 ns MD in explicit solvent were performed at 300 Kon five Aβ9-42 oligomers (monomer to pentamer) with the residues 9-16

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extended using the AMBER 99sb–TIP3P force field (Horn and Sticht, 2010).To enhance sampling, the dimer simulation was extended to 200 ns and thetrimer was also subject to one 100 ns MD at 310 K. It is found that thefibril conformation remains stable in the trimer to pentamer with the twoparallel in-register β-sheets and the connecting turn preserved. In contrast,the monomer evolves to metastable β-hairpins spanning Phe19–Val24 andIle31–Val36, and the dimer undergoes large conformational changes in itsC-terminus. The key feature of the dimer conformation is a rotation of theC-terminal β-strand toward its N-terminal β-strand for one chain resultingin an antiparallel β-sheet, and the high mobility and more disorderedcharacter of the second chain. This conformational change is characterizedby a hydrophobic core centered on Phe19 of CHC. Those MD also revealthat the larger oligomers are stabilized by interchain Asp23–Lys28 saltbridges, whereas an Asp23–Asn27 interaction is found in the dimer.

These two simulations in explicit solvent, although using different pro-tein and water force fields, suggest that the population of the aggregation-prone N∗ structures drastically increases from dimers to trimers. Thisis apparently consistent with recent experiments showing that toxicityincreases upon oligomerization (tetramer > trimer > dimer), and trimersand tetramers are almost as active as preformed fibrils in the nucleation offibril formation (Ono et al., 2009). However, the fact that the Aβ trimers topentamers remain in the vicinity of the starting fibril state within 100 ns isstrong evidence of insufficient MD sampling. This is further supported bythree recent simulations.

Using long DMD simulations, differences in oligomer size distributionsand in the role of the C-terminal region in oligomer formation wereobserved between Aβ1-40 and Aβ1-42 (Urbanc et al., 2010). Aβ1-40assembly proceeds through interactions among the CHC regions andintermolecular β-strand formation in the Ala2–Phe4 N-terminal region.In contrast, Aβ1-42 assembly proceeds via intermolecular interactionsinvolving the Ile31–Ala42 C-terminal regions as well as interactionsbetween Ile31–Ala42 and the CHC region. Interestingly, there is nosubstantial increase of β-sheet content from dimers to hexamers, allcontents varying between 14% and 22%. We recall that the β-sheet contentin fibrils amounts to 50%. DMD further indicates that Aβ1-40 dimersand hexamers have indistinguishable intramolecular contact maps and

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tertiary structures, whereas the Aβ1-42 transition from dimers to hexamersis accompanied by a partial loss of intramolecular contacts within the CHC.Overall, the formation of low molecular weight (LMW) aggregates may notbe accompanied by major structural changes of individual peptides.

Recent all-atom implicit solvent REMD simulations on Aβ10-42monomers, dimers, and tetramers used 24 replicas distributed linearlybetween 300 and 530 K with an increment of 10 K (Kim et al., 2010).The cumulative time for each system reaches about 100 µs. Despite theforce field used over-estimating the α-helix content, the conformationalensembles of dimers and tetramers are found to be very similar, but sharplydiffer from those sampled by monomers. The key structural difference ofoligomers is the formation of β-structure with the loss of intrapeptideinteractions and α-helix structure. The oligomerization interface is largelyconfined to the region 10-23 with an approximately equal number ofparallel and antiparallel contacts, suggesting that Aβ10-42 tetramers havestill to undergo significant structure reorganization en route to fibrilformation.

Finally, the structures of Aβ17-42 trimers were explored by REMD–OPEP simulations starting from the fibril structure. The simulations used22 replicas between 280 and 700 K, each for 1200 ns (Chebaro et al., 2012).Excluding the first 400 ns for analysis, the secondary structure compositionand free energy do not change using block analysis, indicating goodconvergence of the simulations. At 300 K, we find that the averaged α-helicaland β-sheet contents amount to 8% and 6%, and the population of turnand coil reaches 47% and 36%, respectively. The population of the N∗conformation is very low, of the order of 0.3%, and is thus within statisticalerrors. Each peptide behaves essentially as a compact coil-turn chainwith little regular secondary structures, consistent with DMD simulations(Urbanc et al., 2010). Using a root-mean square deviation (RMSD) cut-offof 3 Å, 35% of all sampled conformations can be described, however, bytwo clusters. The first cluster displays one chain with a β-hairpin spanningresidues Phe17–Leu34, and the other two chains with a β–α–β-turn–β

motif. In this motif, the α-helix spans residues Glu22–Lys27, the turn spansGly37–Glys38, and the β-strand signal is rather weak elsewhere (Fig. 4.1c).The second cluster is more disordered with an interpeptide antiparallelβ-sheet spanning the CHC region and residues Ile31–Leu34, an α-helix

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spanning Ala21–Asn26, and turns at positions Gly37–Gly38 (Fig. 4.1d).Overall, these extensive simulations indicate that the preference for parallelβ-sheet geometries is not populated in the trimer of Aβ17-42.

4.5 Conclusions

Multiple sources of toxicity ranging from small to large aggregates have beenrecently identified. Structural characterization of the amyloid β-proteinoligomers forming early in a complex environment with metal ions,proteins, and membranes is of utmost importance in understanding theircausal roles in AD. Although some biased computer simulations start tointegrate external biological factors (Miller et al., 2010b; Luo et al., 2010;Strodel et al., 2010), we have reviewed all unbiased computational studiestargeting the structures of small Aβ oligomers in aqueous solution. Thesesimulations based on different protein representations and force fieldssuggest that the population of the fibril-competent conformation and thepreference for parallel geometry are almost negligible in Aβ dimer andtrimer, although these species are toxic. Revealing all of the molecularevents from monomers to fibrils, determining whether the toxic species areon pathways, and characterizing their three-dimensional atomic structuresclearly requires a systematic effort to combine existing (or new) in vitrosimulation protocols and force fields so as to discover a new generation ofdrugs.

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[Molecular Medicine and Medicinal Chemistry] Alzheimer's Disease: Insights into Low Molecular Weight...

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