[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-chB6

6From Disordered Amyloid

β-Proteins to Soluble Oligomersand Protofibrils Using Discrete

Molecular Dynamics

Mark Betnel ∗, Nikolay V. Dokholyan† and Brigita Urbanc‡

6.1 Introduction

Over the past two decades, studies of amyloid β-protein (Aβ) folding andassembly in vitro, in vivo, and in silico have proven to be challenging,and a synergistic multidisciplinary research approach that combines bothexperimental and computational techniques has been proposed (Teplowet al., 2006). The purpose of the present review is to elucidate experimentaland computational challenges and then describe the results of theapplication of an efficient discrete molecular dynamics (DMD) combinedwith an intermediate resolution protein model to studies of Aβ folding andassembly.

∗Department of Physics, Boston University, Boston, MA 02215, USA.†Department of Biochemistry and Biophysics, School of Medicine, University of NorthCarolina at Chapel Hill, Chapel Hill, NC 27599, USA.‡Department of Physics, Drexel University, Philadelphia, PA 19104, USA.

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334 M. Betnel, N. V. Dokholyan and B. Urbanc

6.1.1 The role of amyloid β-protein in Alzheimer’s disease

Alzheimer’s disease (AD) and the associated Aβ have received particularlyextensive attention in the medical and basic research communities, dueto the prevalence of AD in the general population (Hebert et al., 2003),the devastating nature of the disease symptoms, and the nature of Aβ

as a paradigmatic example of the class of disordered, amyloidogenic,disease-causing proteins (Walsh and Selkoe, 2004). Hallmarks of ADinclude the occurrence of extracellular amyloid plaques in the cerebralcortex, formation of intracellular neurofibrillary tangles, and substantialneuronal loss. In the early 1990s, the amyloid cascade hypothesis identifiedAβ plaques in the brain as proximate neurotoxins in AD (Hardy andAllsop, 1991; Hardy and Higgins, 1992). Aβ, the dominant proteinaceouscomponent of amyloid plaques (Glenner and Wong, 1984), forms assem-blies that have been shown to be directly, and potently, neurotoxic to cellcultures (Hsia et al., 1999; Walsh et al., 1999; Taylor et al., 2002; Lesnéet al., 2006) and mutations in the Aβ primary structure are associated withfamilial forms of AD (FAD) (Citron et al., 1992; Selkoe, 2001; Hardy, 2003).

During the past two decades, substantial evidence accumulated thatimplicates small soluble Aβ assemblies, termed oligomers, and other pre-fibrillar structures as the most potent neurological toxins (Klein et al.,2001; Kirkitadze et al., 2002). The presence of low molecular weightoligomers strongly correlates with the severity of AD symptoms (Gonget al., 2003; Bitan et al., 2005) and the oligomers were shown to be potentneurotoxins (Bhatia et al., 2000; Walsh et al., 2002; Barghorn et al., 2005;Glabe, 2005). For this reason, much recent attention has been focusedon understanding the mechanisms by which Aβ oligomers are formed,exploring mechanisms by which the oligomers might cause cell death, andinvestigating potential inhibitors that could prevent Aβ assembly into toxicstructures (Klein, 2002a; Rahimi et al., 2008).

Here, we distinguish the following levels of structure and complexity inthe study of Aβ: (i) Aβ folded structures, (ii) low molecular weight, solubleoligomeric assemblies that do not have the cross-β-structure characteristicof amyloid fibrils, and (iii) insoluble, elongated fibrils macroscopic lengthsin micrometer range bearing the hallmark amyloid cross-β-structure.

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From Disordered Amyloid β-Proteins to Soluble Oligomers and Protofibrils 335

6.1.2 In vitro studies of amyloid β-protein folding and assembly

Of the two predominant alloforms that occur in vivo, Aβ40 and Aβ42,the latter has two additional C-terminal residues, I41 and A42. Despite asmall difference in the sequence, Aβ42 aggregates faster than Aβ40 (Seubertet al., 1992; Jarrett et al., 1993a,b; McGowan et al., 2005), forms more toxicassemblies (Klein, 2002b), and is genetically linked to aggressive, early-onsetFAD (Sawamura et al., 2000). Aβ40 was shown to be less toxic than Aβ42in vivo in a transgenic mouse (McGowan et al., 2005) and a Drosophilamodel (Iijima et al., 2004), and less toxic in vitro as measured by an assayfor mitochondrial activity (Dahlgren et al., 2002). A recent study by Maitiet al. (2010) compared Aβ40 and Aβ42 toxicities using assays that measuremitochondrial activity, membrane integrity, and DNA fragmentation athigh peptide concentrations of 10 µM, confirming the significant toxicitydifferences between the two alloforms in cell cultures.

In vitro, both Aβ40 and Aβ42 adopt a predominantly α-helical structurein a membrane-mimicking environment (Coles et al., 1998; Crescenziet al., 2002). In α-helix-forming conditions, solution nuclear magneticresonance (NMR) studies identified a relatively common helix–turn–helix motif, with the two helices at approximately residues S8–G25 andK28–G38, and the turn spanning G25–K28 (Janek et al., 2001; Crescenziet al., 2002). In contrast, a collapsed coil structure was identified in anaqueous solution (Zhang et al., 2000). The presence of trifluoroethanol orhexafluoroisopropanol in water was shown to induce Aβ to fold into theα-helical structure in a concentration-dependent way (Barrow et al., 1992;Sticht et al., 1995; Fezoui and Teplow, 2002). Aβ is thus characterized bya high degree of flexibility, with preferred conformational states heavilydependent on the specific solvent conditions (Barrow et al., 1992).

Lazo et al. (2005) showed that the region A21-A30 shared by bothAβ40 and Aβ42 was strongly resistant to protease degradation in bothalloforms, and was thus postulated to represent a nucleus of full-lengthAβ folding. Solution NMR study of the decapeptide fragment Aβ(21-30)itself showed that Aβ(21-30) folds into loop-like structures stabilized bythe effective hydrophobic attraction between V24 and the butyl portion ofK28 (Lazo et al., 2005). The stability of the Aβ(21-30) fold was found to be

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compromised in the decapeptide mutants relevant to FAD in a mutation-specific way (Grant et al., 2007). Several differences in Aβ40 vs. Aβ42 foldingwere observed in vitro. Aβ42 showed an increased protease resistance at theC-terminal region relative to Aβ40, suggesting that Aβ42 but not Aβ40 hada structured C-terminal region (Lazo et al., 2005). Additional independentstudies confirmed that the C-terminal region in Aβ40 monomers was moreflexible than in Aβ42 monomers (Murakami et al., 2005; Yan and Wang,2006; Sgourakis et al., 2007).

Prior to or during the assembly process, Aβ is hypothesized to undergoa conformational transition from the α-helix-rich structure, characteristicof a membrane environment, to the β-sheet-rich structure, characteristicof a fibril. Several studies aimed to identify the protein regions responsiblefor this conformation transition. A D-amino acid analogue of Aβ42 wasused to show that destabilizing the first helix of the helix–turn–helix motifgenerated an α-to-β transition and the formation of fibrils (Janek et al.,2001), and circular dichroism (CD) and NMR studies reported a transitionfrom an unfolded to β-sheet-rich structure in the regions E3–H12and F20–E22 when conditions were altered from non-amyloidogenic toamyloidogenic (Lim et al., 2007). A survey of solvent conditions revealedthat dynamic control of β-sheet content at the C-terminus could beachieved by altering the polar nature of the solvent, with apolar, membrane-like solvents inducing α-helical structure and polar, water-like solventsinducing β-sheet conformations (Tomaselli et al., 2006). Initial studiesof the temperature dependence of the secondary structure of Aβ40 inaqueous solution demonstrated that the β-strand propensity increased withtemperature; however, it was not clear whether this increase in the β-strandpropensity occurred in Aβ40 monomers or was induced by Aβ40 dimerformation (Gursky and Aleshkov, 2000). Using CD spectroscopy on bothAβ40 and Aβ42 in aqueous solution, Lim et al. (2007) demonstrated thatthe average β-strand structure increased with temperature, whereas Aβ

peptides remained monomeric. Aβ42 monomers were reported to havea slightly higher amount of the average β-strand structure than Aβ40monomers, demonstrating additional differences between Aβ40 and Aβ42folded structures.

Various studies have explored Aβ assembly into oligomeric structures.Chromy et al. (2003) found that Aβ42 self-assembled into globular

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oligomers of various orders ranging from trimers to 24-mers that weretoxic to PC-12 cells. Both Aβ40 and Aβ42 oligomers were efficiently cross-linked, and their relative abundances quantified, by using the techniqueof photo-induced cross-linking of unmodified proteins (PICUP) (Bitanet al., 2003a; Bitan, 2006). Using PICUP followed by sodium dodecylsulfate-polyacrylamide gel electrophoreses (SDS-PAGE), Aβ40 and Aβ42were shown to oligomerize through distinct pathways (Bitan et al., 2003a),a notion recently quantified by an independent method using ion mobilitycoupled with mass spectrometry (Bernstein et al., 2009). Bitan et al. (2003a)showed that Aβ40 existed in an equilibrium of decreasing abundancesof monomers, dimers, trimers, and tetramers, whereas Aβ42 displayedmultimodal oligomer size distribution with increased abundances ofpentamers and hexamers, which were termed “paranuclei,” and decamersand dodecamers that were formed by self-assembly of two paranuclei. Ina follow-up study, a PICUP/SDS-PAGE protocol was used to evaluate sys-tematically the oligomerization of 34 physiologically relevant Aβ alloforms,including those containing FAD-linked amino acid substitutions, naturallyoccurring N-terminal truncations, and other modifications (Bitan et al.,2003c). Aβ40 oligomerization was particularly sensitive to substitutionsof E22 or D23 and to truncation of the N-terminus, whereas Aβ42oligomerization was largely unaffected by substitutions of E22 or D23 orby N-terminal truncations, indicating that alloform-specific regions andresidues control Aβ oligomer formation (Bitan et al., 2003c). A subsequentstudy by Bitan et al. (2003b) demonstrated that oxidation of M35 inAβ42 inhibited formation of paranuclei, resulting in a Aβ40-like oligomerdistribution.

The isolation of significant amounts of unmodified Aβ oligomersof a specific order that would enable systematic experimental study ofstructures has proven to be very challenging. However, recently, cross-linked, covalently bonded Aβ40 monomers, dimers, trimers, and tetramerswere successfully examined for average secondary structure and cell culturetoxicity (Ono et al., 2009). The toxicity of these cross-linked Aβ40 oligomersstrongly and non-linearly depended on the oligomer order with tetramersshowing the highest degree of toxicity (Ono et al., 2009).

Although this significant body of in vitro work sheds much light onthe Aβ folding and oligomer formation, their three-dimensional structure

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and details of the interactions driving folding and assembly cannot bedetermined experimentally due to limitations imposed by biophysicaltechniques, the high sensitivity of Aβ structure to external conditions, theheterogeneous nature of Aβ oligomers, and their relatively short lifetime.

6.1.3 All-atom molecular dynamics studies of amyloidβ-protein

All-atom molecular dynamics (MD) in explicit solvent provides the highestdegree of structural information on protein folding and assembly as itinvolves modeling of all protein atoms as well as solvent atoms. Here, abrief overview of all-atom MD applications to Aβ folding and assemblyis given. Most all-atom MD approaches targeted the fragment Aβ(16-22)because it forms fibrils and includes the central hydrophobic cluster thatis believed to be critically involved in aggregation and it is short enoughto be considered for all-atom MD. Aβ(16-22) assembly was studied byKlimov and Thirumalai (2003), who found that Aβ(16-22) monomersadopt compact random-coil or extended β-strand structures prior toassembly into antiparallel β-sheet-like trimers that involves formationof α-helical intermediates. In contrast, Santini et al. (2004) did notobserve formation of α-helical intermediates but found both a paralleland antiparallel arrangement of Aβ(16-22) in assemblies. The tendencyof Aβ(16-22) to assemble into β-sheet structures with both parallel andanti-parallel arrangements (Favrin et al., 2004) was also observed byGnanakaran et al. (2006).

A number of computational studies explored the protease-resistantdecapepetide fragment Aβ(21-30), finding an ensemble of structures witha bend motif spanning V24–K28 and stabilized by a variety of mechanisms,including hydrogen bond networks involving the side-chain of D23 and theamide hydrogens of G25, S26, N27, and K28 (Baumketner et al., 2006b),a salt bridge between E22 and K28 (Cruz et al., 2005; Baumketner et al.,2006b), and hydrophobic packing of V24 and K28 (Cruz et al., 2005).Cruz et al. (2005) noted a high sensitivity of the conformation to solventconditions. Fawzi et al. (2008), however, emphasized the variability ofthe ensemble of Aβ(21-30) conformations, finding the majority of thepopulation to be completely unstructured, with a minority population

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showing a bend motif with the turn at V24/G25 and a salt bridge fromD23 to K28.

All-atom MD studies of the longer fragment Aβ(10-35) identifiedrelatively large energy barriers to interconversion between α-helical,collapsed coil, and β-sheet conformations (Straub et al., 2002), a significantdegree of ordering of water molecules in the first solvation shell aroundthe peptide and its Dutch variant (E22Q), and stronger water–peptideinteractions for the wild-type (WT) than the mutant, suggesting that theWT structure may be partially stabilized by the water (Massi and Straub,2002). Han and Wu (2005) found a stable strand–loop–strand Aβ(10-35)structure that is topologically similar to that found in Aβ fibrils, promptingthem to suggest that the structure is an important intermediate bindingstructure for fibril growth. Low-order Aβ(10-35) oligomers (monomersto tetramers) were observed by all-atom MD in implicit solvent usingenhanced sampling techniques (Jang and Shin, 2008).

A full-length Aβ folding study using replica exchange MD in implicitsolvent found a greater flexibility of the C-terminus of Aβ40 over that ofAβ42 (Sgourakis et al., 2007). A variety of distinct folded Aβ42 conformerswas identified by Baumketner et al. (2006a). Alloform-specific differencesin the free energy surfaces between Aβ40 and Aβ42, with broad basinsof α-helical and β-stranded structure, were also found (Yang and Teplow,2008). In studies of Aβ with lipids, the full-length Aβ has been shown tointeract with model lipid bilayers, forming α-helical structures within thebilayer (Xu et al., 2005), and aligning parallel to the surface of the layerwithout a significant change in secondary structure, indicating that thebilayer is not able to fully order Aβ monomers, but that rather multiplemonomers may be necessary to facilitate the assembly and the emergenceof Aβ fibrillar order (Davis and Berkowitz, 2009).

6.2 Why Discrete Molecular Dynamics?

It is intriguing that a relatively small difference in the sequence betweenAβ40 and Aβ42 (5%) should result in significantly different oligomeriza-tion pathways at a relatively early stage of assembly (Bitan et al., 2003a).As discussed above, structural differences occur already at the stage of

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monomer folding. To be able to draw correlations between Aβ40 andAβ42 structural characteristics and the resulting differential Aβ40 or Aβ42induced toxicities, it is useful to derive Aβ40 and Aβ42 oligomer structuresand characterize structural differences between them. Here we are facedwith two major computational challenges: (i) to derive oligomer structuresas they form in vivo starting from monomeric, spatially separated, andnon-interacting peptides and (ii) to obtain a number of oligomer structuresthat is large enough to characterize the structures in a statistically reliableway. Neither of these two challenges can be tackled by using all-atom MDin explicit solvent.

The application of the efficient DMD with square-well potentials,coarse-grained protein models, and implicit solvent helps to reduce thenumber of degrees of freedom that need to be handled computation-ally (Dokholyan et al., 1998, 2000; Urbanc et al., 2006). Due to thesesimplifications, clear validation criteria that helps to ensure the biologicalrelevance of the DMD approach need to be used. The DMD approachdescribed here is characterized by two implicit solvent parameters thatdepend on the solvent conditions (Urbanc et al., 2006). The validation ofthe approach was achieved through the adjustment of the implicit solventparameter values to obtain the best description of the in vitro observed Aβ40and Aβ42 oligomer size distribution differences. Despite simplifications,assembly studies in general are still computationally demanding and requirethat high peptide concentrations, typically 10- or 100-fold higher than thoseused in vitro, are employed to facilitate the assembly and provide a sufficientnumber of biologically relevant assembly structures (Urbanc et al., 2004b;Pellarin and Caflisch, 2006; Cheon et al., 2007; Pellarin et al., 2007, 2010).

Combining DMD with a four-bead protein model and backbonehydrogen bonding, Ding et al. (2003) showed that a polyalanine chainunderwent a temperature-driven conformational change from an α-helicalthrough a β-sheet to a random coil monomer conformation withoutany a priori knowledge of the native structure. Such a conformationchange is believed to happen in Aβ when released from the membraneenvironment, where it presumably adopts an α-helical conformation (Coleset al., 1998; Crescenzi et al., 2002), to an aqueous environment, where itadopts a collapsed coil conformation (Zhang et al., 2000). Moreover, thisconformational change was postulated to be the key event preceding Aβ

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assembly (Zagorski and Barrow, 1992; Soto et al., 1995; Tomaselli et al.,2006). Thus, the DMD approach with the four-bead model seems to havethe correct ingredients to be biologically relevant and is efficient enoughfor studies of Aβ oligomerization.

6.3 Aβ40 vs. Aβ42 Folding Studied by DiscreteMolecular Dynamics

Folding of full-length Aβ40 and Aβ42 monomers was studied using DMD,a four-bead protein model, implicit solvent, backbone hydrogen bonding,and amino-acid-specific interactions due to the hydrophobic/hydrophiliceffect. Folding of both peptides was seen to begin at the C-terminus withthe emergence of the β-strand structure, and then to proceed towardthe N-terminus. Even in this reduced model, differences could already beseen in the folding stage for these monomers, despite the small differencebetween the two alloforms. At long simulation times, a turn structurecentered at residues G37–G38 emerged in Aβ42, but not in Aβ40. Therewere significant differences in the β-strand propensities in the N-terminalregion. Aβ-strand at A2–F4 was present in only Aβ40 and was laterconfirmed by a subsequent study by Lam et al. (2008), a study that alsoshowed an additional turn region in Aβ42 centered at G9 and flankedby two short regions with high β-strand propensities. These N-terminaldifferences between Aβ40 and Aβ42 monomer structures were not directlyobserved in vitro. In contrast, the in silico observed structural differencesat the C-termini were consistent with findings by Lazo et al. (2005)who used limited proteolysis, mass spectroscopy, and solution NMR tofind a structure at the C-terminus of Aβ42 but not Aβ40 monomer,by Murakami et al. (2005) who used electron spin resonance to find aC-terminal β-sheet region core in Aβ42, by Yan and Wang (2006) whoused NMR to find the C-terminus of Aβ42 monomers more rigid thanthat of Aβ40 monomers, and by Sgourakis et al. (2007) who used NMRconstraints and performed all-atom replica exchange MD simulations tofind that Aβ42 monomers are more structured at the C-terminus than Aβ40monomers.

A later study using the DMD method, four-bead protein model, implicitsolvent, backbone hydrogen bonding, and amino-acid-specific hydropathic

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and effective electrostatic interactions that take into account the solventscreening effect showed that both alloforms undergo conformationaltransitions from collapsed coil to β-strand to more extended structures withincreasing temperature and also found that simulated Aβ42 increased its β-strand content at a faster rate than did Aβ40 (Lam et al., 2008). Both resultswere consistent with CD data (Lim et al., 2007). Lam et al. (2008) furtherrecapitulated the finding of a central folding region centered at G25–S26 in both alloforms, as well as the turn at G37–G38 in Aβ42, but notin Aβ40.

6.4 Aβ40 vs. Aβ42 Oligomerization Studied by DiscreteMolecular Dynamics

The four-bead protein model with backbone hydrogen bonding combinedwith DMD developed by Ding et al. (2003) was applied to study Aβ40 andAβ42 monomer folding and dimer formation by Urbanc et al. (2004a).Asymmetric planar β-hairpin monomer conformations with a turncentered at G25–S26 formed prior to a modular assembly into planar β-sheet dimers with all possible combinations of the parallel and antiparallelarrangement of the two β-hairpin-forming regions, K16–V24 and N27–V36 (Urbanc et al., 2004a). Interestingly, the turn centered in that sameregion (G25–S26) was observed in predominantly α-helical Aβ40 and Aβ42monomers in membrane-mimicking environments (Coles et al., 1998;Crescenzi et al., 2002). Although β-hairpin Aβ monomer conformationswere not found to form in either a membrane-mimicking or aqueousenvironment, the β-hairpin Aβ40 monomer conformation was observedupon Aβ40 binding to a dimeric affibody protein ZAβ3 ligand, which wasshown to create a large hydrophobic tunnel-like cavity that was inaccessibleto water and shielded the Aβ40 β-hairpin from the solvent (Hoyer et al.,2008). This experimental observation is consistent with the structuralpredictions of the β-hairpin Aβ monomer structure derived by the four-bead DMD model that takes into account only backbone hydrogen bondingbut neglects the effective hydropathy, which is associated with the presenceof water. The DMD-derived planar β-hairpin Aβ40 and Aβ42 dimers weresubjected to all-atom MD simulations using implicit/explicit solvent tostudy their mechanical stability and compute their free energy (Urbanc

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et al., 2004a). Although mechanically stable, the planar β-hairpin Aβ dimerswere not thermodynamically more stable than the corresponding β-hairpinmonomers and no alloform-specific differences were observed (Urbancet al., 2004a), suggesting that amino-acid-specific interactions betweenside-chains due to effective hydropathy, charge, or both are needed foran adequate description of Aβ40 vs. Aβ42 dimer formation in aqueoussolution.

The amino-acid-specific interactions due to effective hydropathy andcharge of the side-chain group were introduced by Urbanc et al. (2004b,2006). The strength of the effective hydropathy, relative to the potentialenergy of hydrogen bond formation EHB (a unit energy in the model),represents one of the two implicit solvent parameters, EHP . The otherimplicit solvent parameter ECH is associated with the effective electrostaticinteraction strength in the solvent. Both implicit solvent parameters needto be adjusted to the solvent under study. This is particularly importantfor intrinsically disordered proteins, such as Aβ, for which the structureis very sensitive to the changes in solvent conditions. The addition ofamino-acid-specific hydropathic interactions, EHP = 0.3, to the four-beadmodel, an estimate of the correspondence between model temperature andphysiological temperature (T = 0.15), and relatively long simulation runs(10 million time steps) permitted the study of the formation of oligomersof the two alloforms and resulted in significantly different oligomer sizedistributions, consistent with in vitro (Bitan et al., 2003a) findings (Urbancet al., 2004b). Among Aβ40 oligomers, dimers were most abundant,followed by trimers and tetramers, whereas Aβ42 displayed a multimodaldistribution with the first peak centered between dimers and trimers and thesecond peak at pentamers (Urbanc et al., 2004b). The difference betweenAβ40 and Aβ42 oligomer size distributions (unimodal vs. multimodal)is consistent with two distinct mechanisms of oligomerization. TheDMD and the four-bead protein model with amino-acid-specific implicitsolvent were thus shown to capture key alloform-specific differences inoligomerization.

A closer comparison between the DMD-derived oligomer size distri-butions (Urbanc et al., 2004b) and in vitro size distributions obtainedby PICUP/SDS-PAGE (Bitan et al., 2003a) revealed a lack of in silicoAβ42 hexamers as well as an absence of larger Aβ42 oligomers (decamers

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to dodecamers) that were found by PICUP/SDS-PAGE. One possibleexplanation for this mismatch was the absence of effective electrostaticinteractions in the study by Urbanc et al. (2004b). The role of effectiveelectrostatic interactions was addressed in a subsequent study by Yunet al. (2007), where, in addition to the effective hydropathy (EHP = 0.3),the implicit solvent parameter ECH = 0.6 was used instead of ECH = 0.Surprisingly, both Aβ40 and Aβ42 oligomer size distributions were shiftedtowards larger oligomers (Yun et al., 2007). Although, the Aβ40 andAβ42 oligomer size distributions remained distinct, the most abundantoligomers were significantly larger than those observed in vitro. Recently,a better estimate for the physiological temperature (T = 0.13 instead ofT = 0.15) (Lam et al., 2008) and two-fold longer simulation times (20 × 106

instead of 10 × 106) resulted in an Aβ42 oligomer size distribution withboth abundant hexamers and an additional peak centered at dodecamers(Urbanc et al., 2010).

6.5 Structural Differences between Discrete MolecularDynamics-Derived Aβ40 and Aβ42 Oligomers

The purpose of studying oligomer formation in silico is to elucidate thestructure of oligomers that cannot be quantified in vitro due to themetastable, variable, and heterogeneous nature of oligomers. Struc-tural analysis of DMD-derived oligomers demonstrated quasi-spherical,micelle-like structures with hydrophobic regions comprising the core andhydrophilic regions located at the surface (Urbanc et al., 2004b). The averageamount of the β-strand structure in the DMD-derived Aβ oligomers wasincreased relative to Aβ monomers, from ∼12% in Aβ monomers to ∼19%in Aβ dimers and did not increase significantly upon further assembly intotrimers to hexamers (Urbanc et al., 2010). Although Aβ42 monomers anddimers had a slightly higher average β-strand content than Aβ40 monomersand dimers, the difference was not significant.

Despite a lack of any ordered structure, the quasi-spherical oligomersshow some alloform-specific structural characteristics. Because thesequence difference between Aβ40 and Aβ42 is at the C-terminus, structuraldifferences at the C-terminal region would be expected. In contrast, themost pronounced structural differences between in silico Aβ40 and Aβ42oligomers were observed at the N-terminal region (Urbanc et al., 2004b,

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2010). Aβ40 but not Aβ42 oligomers had substantial β-strand propensitiesin the region A2–F4, resulting in a less flexible N-terminal region relative toAβ42 (Urbanc et al., 2004b). Correspondingly, the solvent exposure of theregion involving the five N-terminal amino acids was significantly higherin Aβ42 than in Aβ40. Aβ42 but not Aβ40 showed a turn centered atS8–G9 separating two regions, R5–D7 and Y10–V12, with increased β-strand propensity. Only Aβ42 formed a short β-strand at V39–V40. Thesealloform-specific structural differences between these in silico oligomersneed to be viewed in the context of their overall fluid and flexiblestructure, so that the above structural tendencies are only averages overlarge populations.

Although Aβ40 oligomer formation proceeded predominantly throughinteractions among the central hydrophobic cluster (CHC) regions andthrough β-strand formation in the A2–F4 N-terminal region, Aβ42oligomerization was characterized by intermolecular interactions involvingthe I31–A42 C-terminal regions, as well as between the C-terminalregion and the CHC region. Although dimers and hexamers of Aβ40had indistinguishable tertiary structure, intramolecular contacts in Aβ42hexamers were weaker than in Aβ42 dimers, suggesting stronger bindingof oligomeric structure in the larger Aβ42 oligomers (Urbanc et al., 2010).The observation that small differences in the C-terminal primary sequenceof the two peptides led to significant structural differences, primarily atthe N-terminus, and that these differences can be revealed through coarse-grained protein models is particularly important. That oligomerization ofthe more toxic Aβ42 proceeds through intermolecular interactions with theC-terminus,whereas Aβ40 oligomerization primarily involves the CHC andpartially the N-terminal region may provide a basis for new structure-basedtherapeutic strategies.

6.6 The Effect of the Arctic Mutation on Amyloid β-ProteinFolding and Oligomer Formation Studied by DiscreteMolecular Dynamics

The Arctic mutation (E22G) of Aβ is associated with a severe, early onsetFAD that is believed to be caused by a faster rate of aggregation of the mutantAβ over the WT, even though the point mutation represents a difference ofonly ≈ 2% in sequence. Just as with alloform-specific differences between

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Aβ40 and Aβ42, an important question is whether coarse-grained proteinmodels can capture differences in folding and oligomerization between theWT and mutant forms, despite the close sequence similarity and relativelylow specificity of the amino-acid-modeling approach.

A DMD study using the four-bead protein model, backbone hydrogenbonding, and residue-specific hydropathic and electrostatic interactionsexamined differences in folding between Aβ40 and Aβ42 and their Arcticmutants, [E22G]Aβ40 and [E22G]Aβ42 (Lam et al., 2008). Both WTalloforms showed a β-hairpin in the central region A21-A30, whereas Aβ42showed β-hairpins atV36–A42 and R5–H13 and Aβ40 showed a β-strand atthe N-terminal A2-F4. The Arctic mutation was found to disrupt the centralfolding region at A21-A30 in both alloforms, and the N-terminal β-strandof Aβ40, leading to increased overall β-strand content in [E22G]Aβ40 overAβ40. These changes made the structure of both Arctic mutants moresimilar to that of Aβ42 than to Aβ40.

A recent study by Urbanc et al. (2010), used DMD and the four-bead model to examine oligomerization of Aβ40, Aβ42, and their Arcticmutants, [E22G]Aβ40 and [E22G]Aβ42, in conditions mimicking anaqueous solvent. As discussed above, Aβ42 had a high propensity toform paranuclei (pentameric or hexameric structures) that could self-associate into higher order oligomers. In contrast to Aβ40, [E22G]Aβ40also formed pentamer/hexamer units, consistent with the PICUP/SDS-PAGE findings (Bitan et al., 2003c). However, of the four peptides, onlyAβ42 actually displayed a significant number of higher order oligomers (oforder 10-13) (Urbanc et al., 2010).

The secondary structure propensities at the N-termini of both Arcticmutants resembled more that of Aβ42 rather than that of Aβ40. Aβ42oligomer formation was strongly driven by intermolecular interactionsamong the C-terminal regions, whereas Aβ40 assembly involved inter-actions in the CHC. The two Arctic peptides showed equally importantinteractions among the C-terminal region (CTR) and CHC regions. Only inAβ40 was the region A2-F4 involved in intermolecular interactions. Intra-and intermolecular contacts between different peptide regions in hexamersof the four alloforms showed that [E22G]Aβ40 hexamers were structurallythe most similar to Aβ42 hexamers. As Aβ40 is more abundant in the human

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body than Aβ42, whereas Aβ42 forms more toxic assemblies, the structuralsimilarity between the [E22G]Aβ40 and Aβ42, in both oligomeric andmonomeric states, is highly suggestive of a mechanism for the early onsetof this FAD.

6.7 The Role of Effective Electrostatic Interactions on OligomerFormation Studied by Discrete Molecular Dynamics

Within the four-bead DMD model, effective electrostatic interactionsbetween two charged side-chain atoms that take into account the screeningeffect imposed by aqueous solvent molecules are implemented using adouble attractive/repulsive square-well potential with the interaction rangerR = 7.5 Å and a “soft” interaction range rSR = 6 Å. The energy of theeffective electrostatic interaction, ECH , is tunable and is expected to be inthe range ECH /EHB ∈ [0, 1], with different ECH values corresponding todifferent solvent conditions (Urbanc et al., 2006).

Strong electrostatic interactions (ECH = 0.6) correspond to desolvatedconditions and lead to a shift in the oligomer size distributions of bothAβ40 and Aβ42 to larger oligomers, as discussed above (Yun et al., 2007).However, even small shifts in the electrostatic interaction strength canlead to relatively large changes in the full-length Aβ oligomerizationbehavior. For example, the recent study by Urbanc et al. (2010) examinedthe effect of treating charged hydrophilic residues as pure hydrophiles(ECH = 0, corresponding to a solvent condition in which charge effectsare completely screened by the solvent), or as very weakly charged(ECH = 10−6, corresponding to nearly complete screening of the chargedgroups). The condition ECH = 0 led to the best match between thein silico and in vitro oligomer size distributions for Aβ40, Aβ42, andtheir two Arctic forms, [E22G]Aβ40 and [E22G]Aβ42, with all fourpeptides forming quasi-spherical oligomers of various order. The shiftto a weak electrostatic interaction led to formation of larger oligomerswith elongated shapes, resembling protofibrillar assemblies commonlyobserved by electron microscopy (Urbanc et al., 2010). This evolution wasa consequence of the structure of the spherical oligomers, which had strongexposure to the solvent of charged residues at the N-terminus (D1, E3, R5,

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and D7) and in the A21–A30 turn region (E22, D23, and K28). In some ofthe largest observed Aβ42 protofibril-like oligomers, undecamers, a parallelintermolecular hydrogen bond pattern was observed, consistent with theonset of the in-register cross-β-structure that is characteristic of in vitroand in vivo Aβ fibrils.

This finding is consistent with the hypothesis that there are threedistinct processes occurring sequentially within the Aβ assembly pathway:(i) fast hydrophobic collapse, resulting in a population of mostly sphericaloligomers; (ii) slow desolvation, enabling charged residues to interactelectrostatically, inducing the formation of elongated protofibrils; and(iii) very slow emergence of parallel in-register intermolecular hydrogenbonding associated with the cross-β-structure of the amyloid fibril (Urbancet al., 2010).

6.8 Discussion

This review provides an outline of the computational approach that couplesDMD, four-bead protein model, and implicit solvent with amino-acid-specific interactions due to effective hydropathy and charge and its applica-tion to study folding and oligomer formation of full-length Aβ relevant toAD. The modeling approach is based on an adjustment of the implicitsolvent parameters, EHP and ECH , associated with effective hydropathyand charge, to the solvent conditions under study. The application ofthe DMD approach to folding and oligomer formation of Aβ40, Aβ42,and the corresponding Arctic peptides, [E22G]Aβ40 and [E22G]Aβ42,demonstrated that the same implicit solvent parameters are able tocapture the distinct oligomer size distribution as observed in vitro usingPICUP/SDS-PAGE (Bitan et al., 2003a,c). DMD-derived oligomers arequasi-spherical molten globule-like structures with hydrophobic regionscomprising the core and hydrophilic N-terminal regions at the surface.A number of oligomers of various order were subjected to structuralanalysis that elucidated a few alloform-specific structural differences. Theaverage amount of the β-structure in in silico monomers and oligomersis consistent with the average β-sheet contents observed in vitro (Kirk-itadze et al., 2001), providing further validation of this computationalapproach.

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The role of the effective electrostatic interactions in Aβ assembly asobserved by the DMD approach is intriguing. The best match to distinctPICUP/SDS-PAGE oligomer size distributions of the four full-length Aβ

peptides was obtained when charged residues were treated as purelyhydrophilic (Urbanc et al., 2010). However, the solid-state NMR andstudies of Aβ40 fibril formation kinetics clearly demonstrate that thesalt bridge between D23 and K28 plays a key role in providing fibrilstability and kinetics (Petkova et al., 2002, 2006; Sciarretta et al., 2005).The fact that the absence of effective electrostatic interactions in thein silico oligomer formation facilitated the best agreement with the in vitrodata was interpreted by consideration of distinct time scales involvedin oligomer vs. fibril formation (Urbanc et al., 2010). Aβ oligomerformation that occurs on a sub-second time scale can be interpreted asa fast hydrophobic collapse during which all of the hydrophilic residues,including the charged ones, remain solvated and hydrogen bonded tosurrounding water, and thus less prone to interact electrostatically andform stable salt bridges. Urbanc et al. (2010) showed that even minorelectrostatic interaction strengths cause further assembly of quasi-sphericaloligomers into elongated protofibrils. It is thus plausible that protofibrilformation is associated with the onset of desolvation of hydrophilicresidues that would allow formation of salt bridges and thus facilitatefurther assembly. Interestingly, a similar view on the hydrophobic collapseand desolvation as two distinct mechanisms that act on different timescales was recently proposed by Javid et al. (2007) who showed that thepartially unfolded state of insulin exposes hydrophobic patches that leadto a drastic increase of the short-range hydrophobic interaction in theaggregation-precursor regime, which is far from conditions where theactual fibril formation takes place, and happens before the entropy ofreleasing the water layers and the attractive short-range hydrogen-bondingforces can provide enough driving force to “dry” out the hydrophilicsurfaces.

Whereas the structure of quasi-spherical Aβ oligomers that areimplicated in the pathogenesis of AD is highly variable even within Aβ

oligomers of the same order, there are a few clear structural differencesbetween Aβ40 and Aβ42 oligomers that might help explain why Aβ42forms larger oligomers than Aβ40 and is potentially more neurotoxic. This

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review thus describes a unique and efficient computational tool that canhelp find the structure–toxicity relationship in the assembly of amyloid-forming proteins and shed light on structural elements that are involved inmediating toxicity.

Acknowledgment

This work was supported by NIH grant AG027818 (Bu).

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

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