A New Alternative "Endosomic" Amyloid Hypothesis?
Amyloid β-protein (Aβ), which is believed to play a critical role at early stages of Alzheimer disease (AD), belongs to the group of natively unfolded proteins. Aβ is known to adopt a collapsed coil folded structure in water, whereas in a co-solvent TFE, which is known to stabilize hydrogen bonding, a pH-dependent α-helical folded structure has been found. Another reason why the pH-dependence is important to understand has to do with the fact that different cellular versus extracellular compartments, where Aβ might be present, have different pH values. Specifically, the extracellular environment is typically characterized by a normal pH = 7.4, while early endosomes (compartments inside the cell) have slightly acidic conditions corresponding to pH = 6. Where the earliest events in Aβ assembly take place, inside the cell, outside the cell, or within a cellular membrane, has been an active research topic for many years. Thus, understanding Aβ folding in different solvent conditions and environments is...  Read more

A New Alternative "Endosomic" Amyloid Hypothesis?
Amyloid β-protein (Aβ), which is believed to play a critical role at early stages of Alzheimer disease (AD), belongs to the group of natively unfolded proteins. Aβ is known to adopt a collapsed coil folded structure in water, whereas in a co-solvent TFE, which is known to stabilize hydrogen bonding, a pH-dependent α-helical folded structure has been found. Another reason why the pH-dependence is important to understand has to do with the fact that different cellular versus extracellular compartments, where Aβ might be present, have different pH values. Specifically, the extracellular environment is typically characterized by a normal pH = 7.4, while early endosomes (compartments inside the cell) have slightly acidic conditions corresponding to pH = 6. Where the earliest events in Aβ assembly take place, inside the cell, outside the cell, or within a cellular membrane, has been an active research topic for many years. Thus, understanding Aβ folding in different solvent conditions and environments is important, because the way the protein folds will directly affect its propensity to assemble as well as impact the structure and function of the assemblies.

Khandogin and Brooks III studied the pH-dependence of Aβ folded structure of two Aβ fragments: Aβ1-28 containing all ionizable residues of full-length Aβ and Aβ10-42, which is more physiologically relevant to the behavior of full-length Aβ. They applied a relatively novel, continuous constant pH molecular dynamics in which protonation equilibrium is achieved by a microscopic coupling at the atomic level, and which allowed Asp, Glu, His, and α-carboxyl residues to titrate. A replica exchange sampling method, spanning a range of temperatures between 298K and 600K, was used to achieve a rapid convergence to the most energetically favorable conformation. The results showed that the α-helix propensity, which on average never exceeded ~10 percent, was in both Aβ1-28 and Aβ10-42 the highest at pH = 2 and the lowest at pH = 6. Importantly, the specific region in Aβ10-42 that was most susceptible to pH was the central hydrophobic cluster (CHC), L17-A21, while the C-terminal region G29-M35 retained the α-helix propensity of the low pH due to a lack of ionizable residues. Importantly, their calculation of the solvent- exposed surface area (SAS) indicated that the CHC residues were the most exposed to the solvent at pH = 6. These data suggested that this region (CHC) is responsible for the conversion from α-helix to a β-strand in TFE co-solvent upon pH increase from 2 to 6. In contrast, the SAS of the C-terminal region of Aβ10-38 was unaffected by pH due to a lack of ionizable residues.

Khandogin and Brooks III also assessed β-turn formation in the region A21-A30. This was reported by many independent groups and seems to be present both in a folded Aβ [1] as well as in the Aβ fibrillar structure [2]. The exact location of the β-turn might depend on the Aβ fragment under study, as in the Aβ21-30 decapeptide a turn at V24-K28 [1] and in Aβ10-35 a turn at Glu22-Asn27 [3] were reported. Khandogin and Brooks III found that the β-turn is most probable at pH 4-8 and that the role of the electrostatic interaction Glu22-Lys28 in Aβ1-28 might be particularly important, in agreement with experimental [1] and computational [4,5] studies on Aβ21-30. However, in more physiologically relevant fragments, such as Aβ10-42, Asp23-Lys28 might play a more prominent role, in particular at the level of fibril formation [2]. Khandogin and Brooks III further showed that strong interaction between Glu22 and Lys28 correlated with destabilization of the α-helical structure in the CHC region. Their examination of residue-specific interactions indicated that at pH = 8, hydrophobic residues of CHC are shielded from the solvent due to effective hydrophobic interactions between the CHC and the C-terminal residues Val40, Ile41, and Ala42. This conclusion is consistent with our simulation study using a DMD approach with a four-bead protein model to examine oligomer formation of Aβ1-40 and Aβ1-42 at physiological conditions (pH ~7.4) [6], which determined the presence of strong contacts between the CHC and the C-terminus in Aβ1-42 but not in Aβ1-40 folded monomers and oligomers.

Interestingly, in our simulations, the strength of electrostatic interactions was minimal, consistent with a lower pH [6]. In fact, including electrostatic interactions involving negatively charged Glu, Asp, and positive charged Lys, Arg, induced formation of larger oligomers in both full-length Aβ peptides [7], consistent with a higher aggregation propensity of Aβ upon increase of pH due to solvent exposure of CHC as observed by Khandogin and Brooks III. Here I want to point out the sequence dependence sensitivity of Aβ structure in both folded and assembly states as observed in our DMD simulations [6,7]. The most surprising result of our simulations of Aβ1-40 and Aβ1-42 folding and oligomer formation was that a difference of two additional amino acids at the C-terminus actually affects the secondary structure and solvent exposure at the N-termini of the two alloforms [6]. Thus, caution is needed when extrapolating data on any Aβ fragment, in particular less physiologically relevant ones, to structure and assembly propensity of full-length peptides.

In conclusion, Khandogin and Brooks III provide novel and much- needed insights into the pH-dependence of Aβ protein folding and assembly, which was found to be hydrophobically driven and most favorable at pH = 6. As pH = 6 is characteristic of early endosomes, these results, combined with existing experimental studies, suggest a new alternative twist on the amyloid-β hypothesis, stating that the initial steps in Aβ assembly may occur inside the cell before secretion into extracellular space.

References:
1. Lazo ND, Grant MA, Condron MC, Rigby AC, Teplow DB. On the nucleation of amyloid beta-protein monomer folding. Protein Sci. 2005 Jun;14(6):1581-96. Abstract

2. Petkova AT, Ishii Y, Balbach JJ, Antzutkin ON, Leapman RD, Delaglio F, Tycko R. A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16742-7. Epub 2002 Dec 12. Abstract

3. Zhang S, Iwata K, Lachenmann MJ, Peng JW, Li S, Stimson ER, Lu Y, Felix AM, Maggio JE, Lee JP. The Alzheimer's peptide a beta adopts a collapsed coil structure in water. J Struct Biol. 2000 Jun;130(2-3):130-41. Abstract

4. Borreguero JM, Urbanc B, Lazo ND, Buldyrev SV, Teplow DB, Stanley HE. Folding events in the 21-30 region of amyloid beta-protein (Abeta) studied in silico. Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6015-20. Epub 2005 Apr 18. Abstract

5. Cruz L, Urbanc B, Borreguero JM, Lazo ND, Teplow DB, Stanley HE. Solvent and mutation effects on the nucleation of amyloid beta-protein folding. Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18258-63. Epub 2005 Dec 9. Abstract

6. Urbanc B, Cruz L, Yun S, Buldyrev SV, Bitan G, Teplow DB, Stanley HE. In silico study of amyloid beta-protein folding and oligomerization. Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17345-50. Epub 2004 Dec 6. Abstract

7. Yun S, Urbanc B, Cruz L, Bitan G, Teplow DB, Stanley HE. Role of electrostatic interactions in amyloid beta-protein (A beta) oligomer formation: a discrete molecular dynamics study. Biophys J. 2007 Jun 1;92(11):4064-77. Epub 2007 Feb 16. Abstract

View all comments by Brigita Urbanc

On Computers, Flies, and Alzheimer Disease
Two recently published papers address the fundamental question of how amyloid proteins form neurotoxic assemblies (see Luheshi et al., 2007 and Cheon et al., 2007). Pat McCaffrey has written an informative and insightful news report that summarizes their key findings and implications. The work reported extends efforts by the ”Cambridge group” (broadly defined, and including those in Firenze, Italy; Busan, Korea; and Jülich, Germany) to explore ”generic” protein folding pathways and their biological consequences. In these latest publications, the group extends the idea of generic protein structures to generic toxicity, meaning that protein assemblies that share structural features also share toxic activity. Importantly, algorithms have been developed that allow prediction of assembly state and neurotoxicity from protein primary structure.

The technical rigor of the two studies is excellent. Thus, within the contexts of the...  Read more

On Computers, Flies, and Alzheimer Disease
Two recently published papers address the fundamental question of how amyloid proteins form neurotoxic assemblies (see Luheshi et al., 2007 and Cheon et al., 2007). Pat McCaffrey has written an informative and insightful news report that summarizes their key findings and implications. The work reported extends efforts by the ”Cambridge group” (broadly defined, and including those in Firenze, Italy; Busan, Korea; and Jülich, Germany) to explore ”generic” protein folding pathways and their biological consequences. In these latest publications, the group extends the idea of generic protein structures to generic toxicity, meaning that protein assemblies that share structural features also share toxic activity. Importantly, algorithms have been developed that allow prediction of assembly state and neurotoxicity from protein primary structure.

The technical rigor of the two studies is excellent. Thus, within the contexts of the experimental systems employed, namely in silico and in vivo (in Drosophila), one may have great confidence in the results. However, now comes the more philosophical and difficult question of meaning. Specifically, how do these results contribute to our understanding of diseases of protein folding?

In this brief discussion, I consider this question and raise a number of others for consideration by the reader. My goal in playing the metaphorical ”Devil's advocate” is to stimulate scientific discourse.

”Meaning” is a nebulous and malleable term for which a definition invariably depends upon the system of evaluation one employs. The goal of the studies of Luheshi et al. and Cheon et al. is to answer the questions of “... the molecular basis of amyloid formation and the nature of the toxic species.” In this context, it is reasonable to ask whether simulating the self-association of Aβ(16-22) or Aβ(25-35) has any relevance (meaning) to Alzheimer disease (AD) or any other disease. Why? Neither peptide is found in vivo. Historically, the former has been a favorite of theorists (including this writer), as its size makes it amenable to in silico study and it forms fibrils in vitro. The latter has been studied since 1990, when the suggestion was made that it was homologous to the tachykinin family of neuropeptides (Yankner et al., 1990). However, the homology relationship was tenuous (as are many when sequence length is so short), authentic tachykinin peptides had no trophic or toxic effects on neurons, and significant evidence supporting the tachykinin connection has not emerged in the subsequent 17 years. Thus, without compelling biological precedent, one must ask what study of these peptides can reveal. For example, are these peptides proxies for holo-Aβ? Clearly, the answer must be ”no,” as the critical determinant of peptide pathogenicity lies at the Aβ C-terminus in the form of the Ile-Ala dipeptide.

Why are people studying what may be irrelevant peptides, and why is such irrelevance not recognized? An answer may come from what, until recently, has been one of the most controversial and contentious fields of modern biology, i.e., prions. The prion theory postulates that the causative agent of a variety of neurodegenerative diseases in animals and humans is composed entirely of protein (no nucleic acid). In the last three decades, the status of this ”protein only” hypothesis in the scientific community has moved from heresy to orthodoxy. However, questions about the scientific appropriateness of this changing perspective have led some, including Laura Manuelidis, to suggest that a re-examination is warranted of ”the objectivity of science and whether it is a myth vanished.” Manuelidis opines that the acceptance of the theory reflects "the peculiarly American sport of betting on popular momentum” (Manuelidis, 2000). A more apropos metaphor, considering that one prion disease is bovine spongiform encephalopathy (“Mad Cow” disease), might have been that of “following the herd.”

Much research on AD could be subject to the same type of criticism. Consider the example of what may be called the "generic” herd. This herd believes that amyloid structure is "generic” because many (most? all?) proteins form amyloids with some common structural organization. Although amyloids, by definition, do share a number of biophysical and spectroscopic features, great structural diversity may be found in the assemblies formed by classical and non-classical amyloid proteins and peptides (e.g., see Sawaya et al., 2007). Importantly, no generic structure outside of the cross-β core of the amyloid fibril has been shown to exist, for obvious reasons. Regions outside of the core, which can be quite extensive in protein, as opposed to peptide amyloids, are likely to influence the biological behavior of the assemblies significantly.

Now, Cheon and colleagues suggest that amyloid formation involves a second generic process, a two-step mechanism of “collapse” of monomers and their subsequent rearrangement into amyloid fibrils. This idea appears to invoke known processes of globular protein folding in the context of amyloid formation, specifically the classical idea of hydrophobic collapse into a molten globule followed by proper arrangement of secondary structure elements to form the native tertiary structure. The idea that some peptides bypass this two-step pathway if they can immediately form hydrogen bonds in their eventual cross-β organization is quite interesting. However, although plausible for short, disordered peptides of the sort studied here, what happens in the common case of natively folded proteins forming amyloid? Here, and as the authors themselves suggest implicitly, factors other than the intrinsic properties of the protein monomer likely moderate amyloid assembly. This increased complexity requires me to question the value of this suggestion of generic mechanisms. Scientists, especially medicinal chemists, need targets. Does a “generic amyloid target” exist? Could a single compound directed at such a target be of value in the treatment of the greater than two score amyloid diseases defined thus far?

Maybe a generic target does exist. In Luheshi et al., studies of the effects of expression of human Aβ42 in Drosophila suggest that protofibril formation correlates with neuronal dysfunction and neurodegeneration. In addition, in a kind of Anfinsen redux (Anfinsen, 1973), an algorithm has been created to predict from primary structure alone the propensity of a protein to form toxic protofibrils. My question: Does the experimental assessment of Aβ-induced locomotor and longevity effects in flies, and its correlation with the toxicity metric, have any relevance to the consideration of Aβ-induced disease in humans? Granted, the same question is sometimes raised as gratuitous criticism of work in a variety of non-human animal models, and it is an easy concern to raise, but that does not diminish the significance of the question.

In closing, it may appear to some that the answers to the questions I have asked are implicit in the construction of the questions themselves. This certainly was not my intention. From a purely academic perspective, I found the publications rigorous, enjoyable to read, and quite thought-provoking. It is the provocation aspect of the experience that operates here, particularly with respect to establishing the meaning of the results and their impact on our shared efforts to understand and treat diseases of aberrant protein folding and assembly.

View all comments by David Teplow

Reply by Leila M. Luheshi, Giorgio Favrin, Damian C. Crowther, Michele Vendruscolo, and Christopher M. Dobson to Teplow Comment
We are pleased to have the opportunity of adding further observations to a recent commentary by David Teplow about the “generic hypothesis” of amyloid fibril formation (1). According to this hypothesis, the ability to form amyloid structures is an inherent property of polypeptide chains, although the propensity to form such structures can vary dramatically with their sequences (2).

This hypothesis is supported by a growing body of experimental evidence that has been summarized in a number of recent reviews (3). The generic nature of amyloid fibrils resides in their core cross-β structure, which is stabilized predominantly by backbone hydrogen bonding interactions (4). It has also been recently discovered that the range of proteins capable of forming toxic oligomers, that may well be precursors to mature amyloid fibrils, is very large and includes those with no known association with disease (5-7). Of course, there are many additional...  Read more

Reply by Leila M. Luheshi, Giorgio Favrin, Damian C. Crowther, Michele Vendruscolo, and Christopher M. Dobson to Teplow Comment
We are pleased to have the opportunity of adding further observations to a recent commentary by David Teplow about the “generic hypothesis” of amyloid fibril formation (1). According to this hypothesis, the ability to form amyloid structures is an inherent property of polypeptide chains, although the propensity to form such structures can vary dramatically with their sequences (2).

This hypothesis is supported by a growing body of experimental evidence that has been summarized in a number of recent reviews (3). The generic nature of amyloid fibrils resides in their core cross-β structure, which is stabilized predominantly by backbone hydrogen bonding interactions (4). It has also been recently discovered that the range of proteins capable of forming toxic oligomers, that may well be precursors to mature amyloid fibrils, is very large and includes those with no known association with disease (5-7). Of course, there are many additional complexities involved in misfolding diseases, and it is also clear that the aggregation of different proteins in vitro does result in fibrils of different morphologies, and the aggregation of different proteins in vivo causes diseases with very different characteristics.

So far the generic hypothesis has provided the inspiration for a number of innovative studies, including the two mentioned by David Teplow (8, 9). We have been astonished by the way that the principles observed in the test tube and in the computer are able to explain the behavior and lifespan of living organisms such as Drosophila. We are therefore optimistic that the insights such studies have given us will lead, in the next few years, to the development of effective therapeutic strategies to combat the debilitating and increasingly prevalent diseases associated with protein misfolding.

References:
1. Dobson CM. Protein misfolding, evolution and disease. Trends Biochem Sci. 1999 Sep;24(9):329-32. Review. No abstract available. Abstract

2. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM. Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature. 2003 Aug 14;424(6950):805-8. Abstract

3. Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 2006;75:333-66. Review. Abstract

4. Knowles, T. P. J. et al. The Role of Inter-Molecular Forces in Defining the Material Properties of Fibrillar Protein Nanostructures. Science (2007) in press.

5. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature. 2002 Apr 4;416(6880):507-11. Abstract

6. Baglioni S, Casamenti F, Bucciantini M, Luheshi LM, Taddei N, Chiti F, Dobson CM, Stefani M. Prefibrillar amyloid aggregates could be generic toxins in higher organisms. J Neurosci. 2006 Aug 2;26(31):8160-7. Abstract

7. Kayed R, Glabe CG. Conformation-dependent anti-amyloid oligomer antibodies. Methods Enzymol. 2006;413:326-44. Abstract

8. Luheshi LM, Tartaglia GG, Brorsson AC, Pawar AP, Watson IE, Chiti F, Vendruscolo M, Lomas DA, Dobson CM, Crowther DC. Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity. PLoS Biol. 2007 Oct 30;5(11):e290. Abstract

9. Cheon M, Chang I, Mohanty S, Luheshi LM, Dobson CM, Vendruscolo M, Favrin G. Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. PLoS Comput Biol. 2007 Sep 14;3(9):1727-38. Abstract

View all comments by Leila Luheshi

Hoyer and coworkers have solved a structure of the amyloid-β peptide in complex with a phage-display selected affibody using NMR spectroscopy. The affibody is responsible for stabilizing the Aβ monomer by inhibiting fibril formation. The Aβ adopts a parallel β-hairpin structure, where the two β-strands consisting of residues 15-22 (strand A) and 30-36 (strand B) form intramolecular hydrogen bonds between each other. Strand A is stabilized by a short strand from the affibody which runs anti-parallel, while strand B is stabilized by a short strand that is parallel to it.

From a large body of data, we know that fibrils exhibit a “cross-β” pattern in x-ray fiber diffraction (1). This is associated with a fundamental structure consisting of extended β-sheet networks in which peptide chains are displayed perpendicular to the fibril axis, while the hydrogen bonding direction of the sheet is parallel to the fibril axis (2,3). Hence, the direction of the hydrogen bonds of a conventional β-hairpin as observed in the current study will not fit the bill.

The authors acknowledge...  Read more

Hoyer and coworkers have solved a structure of the amyloid-β peptide in complex with a phage-display selected affibody using NMR spectroscopy. The affibody is responsible for stabilizing the Aβ monomer by inhibiting fibril formation. The Aβ adopts a parallel β-hairpin structure, where the two β-strands consisting of residues 15-22 (strand A) and 30-36 (strand B) form intramolecular hydrogen bonds between each other. Strand A is stabilized by a short strand from the affibody which runs anti-parallel, while strand B is stabilized by a short strand that is parallel to it.

From a large body of data, we know that fibrils exhibit a “cross-β” pattern in x-ray fiber diffraction (1). This is associated with a fundamental structure consisting of extended β-sheet networks in which peptide chains are displayed perpendicular to the fibril axis, while the hydrogen bonding direction of the sheet is parallel to the fibril axis (2,3). Hence, the direction of the hydrogen bonds of a conventional β-hairpin as observed in the current study will not fit the bill.

The authors acknowledge this fact, and propose that what they observe might be an intermediate structure. In fact, the authors suggest that their structure may amount to the toxic soluble oligomers associated with AD. If this is true, the strands A and B will have to rotate 90 degrees about their axis in order to form the in-register parallel β structure that is likely to form the fibrils. While one cannot rule out this possibility, one must be cautious with such interpretations. It is equally possible that the affibody might be responsible for forming a stable, mixed β-sheet structure. In any event, the affibodies can be useful for passive immunotherapy via the peripheral sink theory (4), as they bind Aβ monomers. Hence, this is an interesting study and the main conclusions require further validation.

References:
1. Sunde M, Blake C. The structure of amyloid fibrils by electron microscopy and X-ray diffraction. Adv Protein Chem. 1997;50:123-59. Abstract

2. Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP, Tycko R. Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils. Science. 2005 Jan 14;307(5707):262-5. Abstract

3. Guo JT, Wetzel R, Xu Y. Molecular modeling of the core of Abeta amyloid fibrils. Proteins. 2004 Nov 1;57(2):357-64. Abstract

4. Solomon B. Intravenous immunoglobulin and Alzheimer's disease immunotherapy. Curr Opin Mol Ther. 2007 Feb;9(1):79-85. Abstract

View all comments by Chris Dealwis

Capturing Aβ Using Engineered Affinity Proteins
Alzheimer disease (AD) is associated with the amyloid-β protein (Aβ) which assembles into toxic oligomers, protofibrils, and fibrils, and is the major component of amyloid plaques in the AD brain. Substantial evidence implicates the early stages of Aβ assembly in the onset of the disease. Many different strategies that aim at preventing Aβ molecules from formation of toxic assemblies are currently under investigation.

The present study by Hoyer et al. was motivated by novel therapeutic strategies that explore ways to create a peripheral sink mechanism by administering an Aβ binding molecule, a ligand, with the capacity to reduce Aβ in the central nervous system by channeling it into the plasma. As the Aβ1-40 binding molecule, Hoyer et al. proposed to apply an engineered affinity protein (affibody), ZAβ3, based on the Z domain derived from the staphylococcal protein A. In their paper, Hoyer et al. presented 16 different ligands which were previously shown to bind both Aβ1-40 and Aβ1-42 and to form dimers through the...  Read more

Capturing Aβ Using Engineered Affinity Proteins
Alzheimer disease (AD) is associated with the amyloid-β protein (Aβ) which assembles into toxic oligomers, protofibrils, and fibrils, and is the major component of amyloid plaques in the AD brain. Substantial evidence implicates the early stages of Aβ assembly in the onset of the disease. Many different strategies that aim at preventing Aβ molecules from formation of toxic assemblies are currently under investigation.

The present study by Hoyer et al. was motivated by novel therapeutic strategies that explore ways to create a peripheral sink mechanism by administering an Aβ binding molecule, a ligand, with the capacity to reduce Aβ in the central nervous system by channeling it into the plasma. As the Aβ1-40 binding molecule, Hoyer et al. proposed to apply an engineered affinity protein (affibody), ZAβ3, based on the Z domain derived from the staphylococcal protein A. In their paper, Hoyer et al. presented 16 different ligands which were previously shown to bind both Aβ1-40 and Aβ1-42 and to form dimers through the intermolecular disulfide bonding between Cys28. Using a combination of powerful methods—isothermal titration calorimetry, circular dichroism, and heteronuclear single quantum correlation (HSQC) NMR spectroscopy—Hoyer et al. derived detailed structural information on the observed ZAβ3:Aβ1-40 complex.

Dimeric ZAβ3 was shown to bind monomeric Aβ1-40 with 1:1 stoichiometry. Binding of ZAβ3 was coupled to folding of Aβ1-40 and the ligand itself. Aβ1-40 was shown to undergo a conformational transition into a β-hairpin conformation upon binding. ZAβ3 dimer, composed of two β-strands and four α-helices wrapping around the Aβ1-40 monomer, created a large hydrophobic tunnel-like cavity. Aβ1-40 within this cavity adopted a β-hairpin with two strands, 17-23 and 30-36, connected by intramolecular backbone hydrogen bonds. Importantly, the cavity of ZAβ3 dimer was inaccessible to water, and the nonpolar faces of the Aβ1-40 β-hairpin inside the cavity were thus mostly shielded from the solvent. The N-terminal region of Aβ1-40, 1-15, and the terminal regions 1-13 and 57-58 of both ZAβ3 subunits were not well defined by the NMR constraints, implying lack of any ordered structure in these regions.

The significance of the ZAβ3 ligand as an inhibitor of Aβ1-40 assembly is that it binds to the hydrophobic part of Aβ1-40 and thereby disrupts Aβ’s potential for further β-sheet extension. To make that possible, the ZAβ3 ligand was engineered such that the two solvent-exposed short β-strands of the ZAβ3 dimer have little propensity to form either α-helical or β-sheet structure.

Hoyer et al. discussed the β-hairpin monomer conformation of Aβ1-40 in relation to the conformation of individual Aβ1-40 molecules within a fibril as proposed in a fibril model by Petkova et al. (1). While the hook-like structure of the β-hairpin monomer resembles the hook-like structure in the fibril model, the hydrogen bonding in the latter is rotated by a right angle with respect to the former. Such a rotation requires a significant structural change as it involves the breaking of intramolecular hydrogen bonds (to destabilize the β-hairpin monomer) and the formation of new intermolecular hydrogen bonds between neighboring Aβ1-40 molecules.

Hoyer et al. suggest that the β-hairpin monomer conformation may be a key conformation for fibril formation to take place. Here, I will give some insights on the significance of the β-hairpin Aβ1-40 conformation from the point of view of a computational physicist. Even though the β-hairpin conformation of Aβ1-40 monomer may be accessible, it may not be energetically favorable under normal aqueous conditions. On the contrary, in aqueous solutions Aβ1-40 monomer adopts a collapsed-coil conformation with less that 15 percent of total secondary structure (2,3). The interactions driving the folding into a collapsed-coil conformation are primarily due to the effective hydrophobicity as also corroborated by computational studies (4). The fact that Hoyer et al. observed a β-hairpin monomer structure of Aβ1-40 within the ZAβ3:Aβ1-40 complex is very much to do with the particular local environment associated with Aβ1-40 folding. The fact that the observed cavity formed by the ZAβ3 dimer is water inaccessible is critical for understanding Aβ1-40 β-hairpin formation. With no water present, there is no hydrophobic effect, so Aβ1-40 folding cannot be driven by the hydrophobic collapse as in the case of aqueous environment. In the absence of the hydrophobic effect, molecular dynamics simulations indeed show that a β-hairpin and other β-sheet monomer structures with up to four β-strands are formed, leading to formation of planar β-sheet dimers (5). This same study also showed that in water, these planar β-sheet monomer and dimer conformations would be equally favorable to both Aβ1-40 and Aβ1-42—a result that is difficult to reconcile with the fact that Aβ1-42 forms larger assemblies faster than does Aβ1-40. While the computational study of Aβ oligomer formation indicated the presence of a turn/loop centered at G25-S26 in both Aβ1-40 and Aβ1-42, with the local structure that resembles the β-hairpin, the intramolecular hydrogen bonding is at best weak, consistent with the importance of effective hydrophobicity in oligomer formation (4). Finally, the presence of the intramolecular hydrogen bonding in a β-hairpin conformation may actually increase the free energy barrier for transition from a monomeric β-hairpin into a fibril-like structure because breaking the hydrogen bonds in a β-hairpin is energetically unfavorable. Thus, it would be energetically more favorable to form a fibril-like structure from collapsed-coil monomer/oligomer conformations, which already possess a turn structure at G25-S26, held together by hydrophobically driven forces rather than intramolecular hydrogen bonds.

In conclusion, Aβ1-40 monomer forming the β-hairpin structure upon dimeric ZAβ3 binding may create a large free energy barrier preventing Aβ1-40 to form toxic assemblies and thus provide a new and exciting therapeutic strategy.

References:
1. Petkova AT, Ishii Y, Balbach JJ, Antzutkin ON, Leapman RD, Delaglio F, Tycko R. A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16742-7. Abstract

2. Zhang S, Iwata K, Lachenmann MJ, Peng JW, Li S, Stimson ER, Lu Y, Felix AM, Maggio JE, Lee JP. The Alzheimer's peptide a beta adopts a collapsed coil structure in water. J Struct Biol. 2000 Jun;130(2-3):130-41. Abstract

3. Kirkitadze MD, Condron MM, Teplow DB. Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis. J Mol Biol. 2001 Oct 5;312(5):1103-19. Abstract

4. Urbanc B, Cruz L, Yun S, Buldyrev SV, Bitan G, Teplow DB, Stanley HE. In silico study of amyloid beta-protein folding and oligomerization. Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17345-50. Abstract

5. Urbanc B, Cruz L, Ding F, Sammond D, Khare S, Buldyrev SV, Stanley HE, Dokholyan NV. Molecular dynamics simulation of amyloid beta dimer formation. Biophys J. 2004 Oct;87(4):2310-21. Abstract

View all comments by Brigita Urbanc