|Home | About | Journals | Submit | Contact Us | Français|
Amyloids can have normal biological functions.1−3 However, amyloidogenic proteins can also form unwanted oligomeric or polymeric aggregates when disturbed from their native functional states. Such aggregates are associated with numerous neurodegenerative diseases, such as diabetes type II,(4) Alzheimer (AD), Parkinson, Huntington, and prion (“mad cow”) diseases, amyotrophic lateral sclerosis, and Down’s syndrome (DS).5−9 Increasing evidence from studies in human, transgenic mice, cultured cells, wild type rodent, and in vitro systems indicates that soluble oligomers of amyloidogenic proteins are both responsible for amyloidosis10,11 and are the toxic agent.12−14 Some data suggest that their final large aggregates can also lead to cytotoxicity.15,16
Ordered aggregates can extend into β-strand enriched fibrils, regardless of their initial native conformational states. Experimental and computational approaches revealed structural details of these organizations. Amyloid structural models were obtained by different methods: spectroscopy,17−23 solid-state NMR (ssNMR),24−29 EPR,30,31 hydrogen/deuterium exchange,15,27,32 cryo-electron microscopy (cryo-EM) and electron microscopy (EM),33−36 X-ray fiber diffraction and X-ray diffraction,37−41 and peptide design.(42) Apart from a handful of peptides that were successfully crystallized providing insight into the ordered amyloid arrangement,(43) amyloid structural elucidation has been fraught with difficulties. The insolubility of amyloid fibrils has made crystallization and solution NMR virtually impossible. Under these circumstances, ssNMR has been the method of choice for structural determination, providing insight into the nature of β-sheet organization.(44) ssNMR data of Aβ segments coupled with atomistic molecular dynamic simulations have further been useful in addressing the driving forces for targeted associations.45,46
To date, the key question of the mechanism through which amyloids lead to cytotoxity is still a major challenge.47−51 As in folding, it is well established that in amyloid fibrils, nucleation and kinetics depend not only on inherent features such as the amino acid composition, sequence, and length, but also on environmental conditions such as temperature,(52) concentration, pH,(53) metal ions, agitation, and enhanced aggregation on the lipid bilayer surface. At the same time, the self-assembly mechanism that leads to ordered fibril formation is not fully understood. Open questions relate to (1) how the monomeric peptides assemble into oligomers; (2) which segments of a long peptide constitute the recognition motifs and as such play key roles in amyloid fibril formation;(54) (3) how the β-strands arrange relative to one another; (4) are there favored organizations between the β-sheets, and if so (5) what are these and what are the intermolecular interactions between the layers that stabilize these; and, finally, (6) what are the pathways and the intermediate states that are involved in seed and fibril formation. These combine to contribute to one of the most difficult issues that are related to protein aggregation, that is, aggregate polymorphism; the aggregates can have different preferred fibril architectures depending on (even slight) changes in any of these sequence or environmental factors.
Amyloid β peptide (Aβ peptide) has served as a paradigm for studies of amyloid formation and conformations. The full-length Aβ peptide has 40−42 residues when cut from its precursor protein. Aβ aggregates are observed in brain tissues of Alzheimer’s patients,(55) and it is generally believed that Aβ peptide oligomerization is a major mechanism leading to the neuron-cell death.(15) This Review focuses on polymorphic Aβ and other amyloid conformations. It compiles the conformations of the full-length Aβ1−42/Aβ1−40 and its fragments, and presents amyloid assemblies in terms of the energy landscape. In addition, it describes how, via conformational selection and population shift as the primary molecular recognition mechanism,(56) monomeric Aβ states whose shapes are compatible can assemble into organized fibril geometries.56−58 Together, this leads to an overview of the structural variability and the underlying mechanisms of fibril polymorphism. Understanding the mechanisms and the range of structural features of the aggregates are of crucial importance for effective drug design to reduce aggregate formation.
Most of the information on oligomeric assemblies is obtained from the full-length β-amyloid peptide Aβ1−42/Aβ1−40.15,25,59−69 The sequence of the human Aβ1−42 peptide is 1-DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA-42. Both the Aβ1−42 and the shorter Aβ1−40 peptides derive from cleavage of the transmembrane amyloid precursor protein (APP) by β- and γ- secretases.(70) There are variations in proteolytic pathways, and thus different fragments can exist in vivo. The cleavage by α- and γ- secretases leads to Aβ17−42 in the so-called “non-amyloidogenic cleavage”.9,71
Experimentally,(53) different full-length Aβ1−40 conformations are preferred in the fibril at different pH values. Petkova et al.(72) observed that the distinct fibril morphologies reflect the variation in the molecular structure of Aβ1−40 at the protofilament level. As shown in Figure Figure1A,1A, subtle variations in fibril growth conditions such as an agitated or quiescent environment translate into morphological changes of the dominant amyloid structures; these are captured in the amyloid seeds and self-propagate.(72) In a recent comprehensive study of Aβ1−40 fibril polymorphism, using cryo-EM and three-dimensional recognition techniques, Meinhardt et al.(73) have shown that structural persistence and morphological diversity can be observed in Aβ1−40 fibrils grown under the same buffer solutions (Figure (Figure1B).1B). However, with two more residues, the longer Aβ1−42 peptide displays even larger variability than Aβ1−40 in the monomeric conformation and in fibril morphology. The fibril organization (and neurotoxicity) of Aβ1−42 peptides also change when mixing WT M35L and F19G mutants (Figure (Figure11C).(15) Zhang et al.(36) suggested an Aβ1−42 conformation with the C-terminus forming the inside wall of a hollow core and the N-terminus playing a role in the fibrilization.
Polymorphism of full-length Aβ peptides observed in experiments. (A) Transmission electron microscopy (TEM) images of amyloid fibrils formed by the Aβ1−40 peptide, prepared by incubation of Aβ1−40 solutions under ...
In addition to full-length Aβ peptides, a number of in vitro-generated β-amyloid fragments were also observed to form amyloid fibrils. The sequences cover all Aβ peptide regions: the N-terminus, middle, and the C-terminus regions. An early study indicated that Aβ18−28, Aβ16−28, Aβ14−28, Aβ12−28, and Aβ1−28 fragments were sufficient to form amyloids; however, Aβ20−28 did not yield a clear amyloid structure.74,75 Other studies extended the fragment list to include Aβ11−25,76,77 Aβ16−22,(78) Aβ10−23,(79) and Aβ14−23.80,81 Figure Figure2A2A and B provides EM images of the Aβ1−28 and Aβ10−23 amyloids, respectively. For Aβ1−28, an assortment can be seen, including linear, curvilinear, and rosette. The amyloid fibril of Aβ10−23 has a diameter around 3 nm and a length of 500 nm, and for the amyloid fibril of Aβ1−28 the diameter is in the range of 6−8 nm and the length in the range of 120−160 nm. Among these, the Aβ16−22 is one of the most studied fragments by both experimental78,82 and computational approaches.83−90 The sequence of Aβ16−22 contains the KLVFF core. Starting from the KLVFF, Tjernberg et al.(91) systematically synthesized 10 peptides with one more residue on each side (Aβ6+x,30−x, where 0 ≤ x ≤ 9). Under their experimental conditions, apart from three peptides (KLVFF, QKLVFFA, and HQKLVFFE), all peptides formed amyloid fibrils with different morphologies.(91)
Polymorphism of Aβ peptide fragments as observed in EM images. (A) Aβ1−28 fibrils, negatively stained, are periodically stained along some of their edges (indicated by arrows). Fibril ends appear to splay or unfurl. Reprinted with ...
Extension from 1−28 to 1−30, 1−33, 1−36, and 1−39 presented fibril morphologies similar to the full-length Aβ.(92) These Aβ sequences are often used to represent full-length Aβ in studies of amyloid formation and toxicity, particularly those including Aβ25−35 and Aβ10−35, which are believed to be the major responsible species.67,93−99 It appears that Aβ25−35 could be more toxic than Aβ1−42, leading to a rapid lysis of red blood cells(97) and causing more extensive oxidative damage.(100) The short version of the Aβ25−35, Aβ26−33, also forms thin long amyloids with a uniform diameter of 5 nm(101) (Figure (Figure2C).2C). Aβ10−35 has a supramolecular structure similar to the full-length.(67) Using computational approaches, Ma and Nussinov studied the Aβ10−35 fragment as a model to study the full-length Aβ, and were the first to discover the U-turn conformation in Aβ.46,83
The C-terminal region (residues 17−42) is mostly hydrophobic. One study observed that the transmembrane part of Aβ29−42 forms amyloids faster than the full-length one, with the order of Aβ29−42 > Aβ1−42 > Aβ1−32 > Aβ1−28;(102) on the other hand, another study found that this peptide scarcely formed filaments. The few fibril structures that could be visualized had a diameter 2−3 nm and an average length of 50 nm.(79) Hilbich et al.(79) explored X-43 peptides (i.e., peptides with an additional residue at the C-terminus). They found that Aβ4−43, Aβ8−43, Aβ10−43, and Aβ12−43 form more stable, tightly packed structures. They further observed that if the peptides are suspended in aqueous solvents, the N-terminal can also promote amyloid formation: the fibril formed by the Aβ12−43 peptide is less stable than the one formed by the Aβ1−43.(79) Aβ34−42 formed rigid fibrils of varying lengths ranging from 10 to more than 300 nm; the single fibrils were slab-like with no apparent twist and had a width of 9 nm. The assembly of several of these smaller fibrils likely constitutes twisted fibers (Figure (Figure22C).44,101 Generally, it seems that amyloid fibrils formed from C-terminal regions are less smooth than those from the N-terminal region. Apart from the Aβ34−42 fragment, the nonsmooth features of the p3 peptide fibrils (Aβ17−40/Aβ17−42)(103) (Figure (Figure2D)2D) further support the potential structural variability of the C-terminal, which could relate to its hydrophobic nature promoting polymorphism (see section 3.3).
Overall, in addition to fiber diffraction, which over the years has provided a wealth of useful structural information,(104) computational studies of monomeric and oligomeric structures of various fragments have clearly shown structural flexibility and oligomerization complexity, for example, Aβ10−35,83,105 Aβ16−35,83,106 Aβ17−42,107,108 Aβ10−35 monomers,109−114 Aβ21−30,115−118 Aβ13−23,(91) Aβ1−28,119,120 and Aβ14−23.(91)
The structural regularity of amyloids is not as good as that of the highly ordered crystals; nonetheless, X-ray fibril diffraction has provided important structural features. Examples of fibril structure variability of Aβ11−25 and the full-length Aβ1−40 obtained by X-ray diffraction are shown in Figure Figure3A.3A. Early diffractions revealed that all amyloids, regardless of their sequences and secondary structures in the native folded states, convert to β-sheets. The β-strand backbone orientation is perpendicular to the fibril axis, which is termed cross-β structure (Figure (Figure3C).3C). The distance between the β-strands is ~0.5 nm, permitting favorable backbone hydrogen bonds, and the layers are usually separated by ~1 nm allowing closely packed β-sheet association (Figure (Figure33B).(77) With these basic features unchanged, the amyloid polymorphism derives from the way the β-strands associate into fibrils. There are three major structural features that may decide the overall amyloid fibril morphologies: (1) differences in backbone orientation; (2) differences in backbone conformation; and (3) differences in the way in which the oligomers, with almost identical structure, associate. The combination of these three factors can give rise to an enormous variation in conformational detail and, consequently, in seed oligomer and fibril morphology. This section demonstrates and discusses these structural features in detail.
(A) EM images and X-ray diffraction patterns of cross-β ribbons for Aβ11−25 (I) and (III) and Aβ1−40 (II) and (IV). The measured widths of the fibrils are ~5 nm for Aβ11−25 and ~7 ...
The detailed structural variations of tightly packed (“steric-zipper”) segments of amyloid-forming peptides(43) and the amyloid backbone associations can be applied to other short peptides like Aβ fragments. In principle, there are thousands of possible patterns of intra- and inter-residue amyloid fibril backbone strand organizations for in-register and out-of-register interactions. Figure Figure44 highlights several representative in-register alignments with double-layered β-strand segment arrangements within and between sheets. Figure Figure4A−D4A−D presents organizations where each layer has parallel strands; within these, parallel orientations between the two layers are illustrated in Figure Figure4A4A and B and antiparallel orientations between the two layers of the segment in Figure Figure4C4C and D. Figure Figure4E−H4E−H presents antiparallel arrangements in each layer, with Figure Figure4E−G4E−G illustrating parallel orientation between the two layers and Figure Figure5H5H an antiparallel orientation. Other possible models of backbone organization can consist of parallel dimers (or trimers, or variable oligomer sizes, etc.) associated in an antiparallel orientation.
Potential architectures of strand organizations in amyloid fibrils. Interactions (in dotted lines) are shown between the first layer (blue) and the second layer (red) for (A) parallel to parallel, face-to-face orientation; (B) parallel to parallel, face-to-back ...
Illustration of Aβ25−35 in parallel (A) and antiparallel (B) arrangements. The sticks are peptide backbone, and the balls are Ile residues. Parts (C) and (D) illustrate the U-turn structures of Ma−Nussinov−Tycko (ref ( ...
Short segments are more convenient systems, because their smaller sizes make them easier to study experimentally and computationally; at the same time, the basic principles are likely to be unchanged. Most studied Aβ segments25,44,64−68,74−76,78−80,91−96,101 are organized in an in-register, parallel arrangement; however, some present a preferred antiparallel organization (enumerated in Table Table1).1). While only some of the potential organizations have been observed, others may also exist, except that their populations may be too low to be observed by experiment.
Features of the Full-Length Aβ Amyloid, Aβ Segments, and β2M (K3)
Short fragments (less than 15 residues length) tend to have antiparallel β-strands. On the basis of X-ray diffraction and Ala16Aβ1−28 mutant substitution comparison, the β-strands’ orientation of Aβ1−28 fibrils was proposed to be all-antiparallel (as in Figure Figure44H).(75) As shown in Figure Figure3B,3B, the Aβ11−25 has antiparallel β-strands within the sheet; however, the sheet−sheet orientation is parallel (as shown in Figure Figure44G).(77) For the Aβ14−23, the antiparallel conformation has also been proposed from NMR information.(80) For Aβ16−22, which is one of the most studied fragments, both experimental and computational studies suggest antiparallel organization.83−90 In the C-terminal region, Aβ34−42 was also shown by ssNMR to be antiparallel.(44) IR spectroscopy indicates that amyloid fibrils of Aβ29−42 and Aβ10−23 have antiparallel β-sheet and less ordered structure (loops or β-turns).(79)
Polymorphism can thus arise from different β-strand orientation and side-chain registration. For example, Aβ16−22 can have two stable antiparallel orientations (Figure (Figure4G4G and H)(83) as both have in register side-chain interactions. Recent experiments also revealed an out-of-register, one residue shift in the antiparallel orientation.(82) Aβ25−35, a central Aβ region fragment, may have both parallel (Figure (Figure5A)5A) and antiparallel (Figure (Figure5B)5B) orientations; both arrangements are probably stabilized by hydrophobic interactions.(121) However, the parallel orientation could be more ordered due to a strong steric zipper match of central Ile residues (Figure (Figure5A5A and B); Ile was proposed to promote amyloid formation in general.(122) As indicated in Figure Figure5,5, Aβ25−35 gives rise to different morphologies depending on whether it is oriented in parallel or antiparallel. A similar scenario arises from parallel or antiparallel arrangements of the peptide hIAPP20−29, corresponding to the amyloid of the SNNFGAILSS region of the human islet amylin polypeptide.(123) There, ssNMR measurements presented two different structural forms of amylin fibrils, responsible for type II diabetes.(123)
Full-length and other long Aβ fragments, especially those containing central and C-terminal regions (for example, Aβ10−35), mostly have parallel β-strands orientation. However, the tendencies for a U-turn backbone conformation in the central region provide yet another variable feature for Aβ peptides. The flexibility of the turn region allows the Aβ peptide to adopt slightly different turn types leading to different amyloid morphologies.
Two models of the three-dimensional structures of Aβ1−42/Aβ1−40 fibrils were proposed and verified experimentally.(46) The first, Ma−Nussinov−Tycko model,46,68 presents a double layer structure with residues 10−22 and 30−40 forming β-strands and residues 23−29 a bend or loop. The two β-strands form two separate, in-register, parallel β-sheets, which can interact with one another due to the intervening bend segment (Figure (Figure5C).5C). The second model by Lührs et al.(15) presents a parallel single layer structure of Aβ1−42 fibrils based on hydrogen/deuterium exchange NMR data, combined with side-chain packing constraints from pairwise mutagenesis, ssNMR, and high-resolution cryo-EM (Protein Data Bank code, 2BEG). Observations based on these experiments indicated that residues 1−16 are structurally disordered and residues 17−26 and 31−42 form two β-strands, β1 and β2, respectively, connected by a U-turn spanning four residues, 27−30 (Figure (Figure5D).5D). This model also suggested that parallel oligomers associate in parallel. The difference between the two U-turns is that in the Ma−Nussinov−Tycko model it is tighter, thus allowing the C-terminal residues to interact with up to N-terminal residue Tyr10;46,68 on the other hand, the Lührs’ turn is looser, with Lys28 shifted, and consequently the C-terminal region can only reach residue Leu17.(15)
Conformational differences in the U-turn can be expected to lead to polymorphic sheet registration and β-strand organization at the termini. For the Aβ case, although both models suggest parallel sheets, the two models differ in the U-turn conformation, leading to different extents of disorder in the N-terminal. In Tycko’s model, the U-turn segment extends over 7 residues and in Lührs’ only over 4. As to the N-terminal, the disordered segment in Tycko’s model includes 8 residues, whereas in Lührs’ it is 16, because the N-terminal projects into the solvent much beyond the C-terminal, thus without tight vdW interactions. This also affects the sheet−sheet registration. Thus, polymorphism in parallel Aβ1−42/Aβ1−40 (or fragments that include the U-turn segment and the two β-strands, such as Aβ10−35) appears to be largely governed by the U-turn conformational variability, and an interplay between various factors can also expected to play a role in polymorphism. Consistently, a combined experimental and simulations study revealed varied U-turn shapes forming by different segment sizes: Aβx−42 (x = 29−31).(124) Recent simulations presented variations for Aβ10−40/10−42(125) and Aβ1−42126−128 due to the U-turn conformation.
It is frequently noted in the literature that the amyloid fibril is a supramolecular structure composed of several protofibrils wound around each other mostly in a left-handed twist along their main axis.35,129−137 Different U-turn conformations can affect the twist angles leading to different oligomer and fibril morphologies. Presumably, a difference in the U-turn conformation exhibits a difference in the interstrand angle; a change of even a fraction of a degree can significantly change the twist. The change will not present a right-handed twist, but will present different EM morphologies.
Apart from the large structural variations described above, small changes in side-chain orientation or in the environment (e.g., pH, temperature, concentration, salt, agitation, synthetic versus brain-seeded) may lead β-sheet oligomers to associate in different ways, leading to amyloid polymorphism with similar molecular organizations. This has been illustrated in Aβ1−40 fibril morphologies. Recently, polymorphism of Aβ1−40 at the protofibril level had been obtained by Meinhardt et al.(73) They classified 12 single Aβ1−40 amyloid fibrils differing in the fibril width and twist angles, all forming under the same solution conditions. As shown in Figure Figure6A,6A, the differences derive from the protofibril associations with altered contact areas. One of the distinct features of the polymorphism observed by Meinhardt et al.(73) is that 2-fold symmetry is mostly conserved for all 11 amyloid fibril models (except fibril model 12) constructed from cryo-EM (not shown here). Fibril model 12 has a different symmetry and organization: even though it still consists of two protofibrils, they are offset with respect to the central fibril axis, alternating from the left-handed to the right-handed and with different mechanical properties.(73) A larger deviation from the 2-fold symmetry of Aβ1−40 fibril morphologies has been observed by Paravastu et al.(138) They found two Aβ1−40 fibril morphologies differing in several features: the overall symmetry (2-fold versus 3-fold); the conformation of the non-β-strand (U-turn/loop) segment; and the quaternary contacts (Figure (Figure66B).(138) Recently, Paravastu et al.(139) have shown that the predominant molecular structure in brain-seeded with Aβ1−40 fibrils differs from the structure of purely synthetic fibrils that were previously characterized.68,138 Fandrich et al.(140) defined polymorphism based on variations in (1) the protofilament number, (2) the protofilament arrangement, and (3) protofilament substructures.
Nonetheless, overall, Aβ1−40 fibrils present regular smooth amyloid morphologies. Many smaller fragments, particularly those containing C-terminal residues (like the Aβ17−42 and Aβ34−42 fibrils, Figure Figure2),2), are much more irregular as compared to the full-length Aβ1−40/Aβ1−42. The potential variability of the fibril morphologies for these amyloids is expected to be much more diverse.
Polymorphism in amyloid fibrils has also been observed for other proteins, for both designed model systems and disease-related proteins. For example, Verel et al.(141) demonstrated polymorphism in a de novo designed peptide model system (Ac-SIRELEARIRELELRIG-NH2), which is expressed by different arrangements of the β-sheets in the oligomer. Depending on the pH, the model peptide can shift the registration of residues and still keep an antiparallel β-sheet organization. Disease-related amyloid polymorphism can be illustrated by the cross-seeding fibrilization of the Q/N-rich protein, which leads to pathological diversity in Huntington disease with the morphologies differing with the segment size.(142)
Among the other species that form amyloid fibrils is β2-microglobulin (β2M), which is a component of type I major histocompatibility antigen. In patients on long-term hemodialysis, β2M can aggregate into amyloid fibers and cause dialysis-related amyloidosis.143−149 The ssNMR structure(150) of a 22 amino acid (Ser20-Lys41) region (the so-called K3) that may have a role in amyloid formation of β2M was investigated using a series of peptides obtained by cleaving with Achromobacter protease I.(151) Interestingly, K3 also shares similar U-turn shape in the β-sheet organization with a parallel orientation.(150) K3 was also shown to form ion-conducting channels in membranes.(51) Other key regions in β2M shown to be important in fibril formation have also been investigated by Radford,152,153 Eisenberg,154−156 and Miranker(157) and their co-workers. β2M segments also exhibit polymorphism. The K3 peptide may form two types (f218 and f210, which has more β-sheet and is thinner and longer) of amyloid fibrils differing in their amino acid contacts between the β-sheets, and both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril’s conformation.(158) Upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation to higher β-sheet content. Full-length β2M has been shown to form at least three different subunit interfaces in the fibril structures assembled from globular tetrameric units.(159) The elegant cryo-EM maps (Figure (Figure7)7) illustrate three-dimensional reconstructions of type A (Figure (Figure7A7A and C) and B (Figure (Figure7B7B and D) forms of the homopolymeric assembly of β2M. The two fibril types have the same underlying organization that differs only in the orientation of the two stacks, either parallel or antiparallel.(159)
Amyloid polymorphism has also been observed for the prion. Yeast prion variants have been identified on the basis of stabilities and phenotypic intensity160−163 relating to prion strains.164−166 Fibril structural polymorphism has been proposed as the culprit in the species barriers167,168 with the amyloid-forming domain in prion filaments exhibiting polymorphic variation.(169) Prion amyloid structural variations have been observed with differences in long-range axial repeats, diameter, and/or the number of protofilaments.(169) Polymorphic prion structures may form due to conformational self-seeding characteristics, kinetics, and stability.(170) Fibrils that are conformationally less stable produce shorter pieces upon fragmentation.(171) The more stable fibril core regions were also identified.(172)
Amyloid polymorphism has also been observed for the islet amyloid polypeptide (IAPP) amylin, by experiment123,173,174 and simulations.(175) Amyloid formation by amylin leads to cell death and is related to type II diabetes. It was demonstrated that conformational polymorphism of IAPP peptides in different microenvironments correlates with cellular toxicity and proteasomal inhibitory activity.123,173,174 The peptide hIAPP20−29, 20NNFGAILSS29, may have two amyloid-like forms, which have distinct structures at the molecular level. One form has parallel β-strands and the other antiparallel, with both stabilized by hydrophobic contacts with similar energy.123,173,174 Hydrophobic interactions can weaken under high hydrostatic pressure. However, fragments 1−19 and 1−29 of IAPP are resistant to pressure, suggesting more densely packed aggregate structures.123,173,174
Amyloid fiber growth involves folding and association via conformational selection of peptide monomers.57,70,176,177 Prediction of the aggregation rate of different Aβ segments may be performed by a model based on the physicochemical properties of the segment.(178) The β-sheet in Aβ segments, while defined by interstrand hydrogen bonding, owes its stability mainly to intra- and intersheet side-chain packing.(179) The presence of intermolecular hydrogen-bonding interactions along the entire length of the peptides further stabilizes the oligomers and is critical in determining the fibril structure.(180) As can be seen from Table Table1,1, some of the Aβ peptide segments adopting β-strand conformations form amyloid fibrils in a parallel orientation and others in an antiparallel orientation. This section examines the properties of these fragments that can affect their preferred architecture such as segment length, hydrophobicity, hydrophilicity, and charge. Table Table11 also summarizes the characteristics of full-length Aβ and its segments and the K3 segment of β2M for which there are ssNMR data.
Different lengths of Aβ segments have different morphologies. As can be seen from Table Table1,1, there is a general trend: longer segments prefer a parallel orientation. Segments with more than 15 residues are organized in parallel; under 15 residues they prefer an antiparallel arrangement probably due to repulsion by the charged termini in the parallel organization, which would need to be overcome. For short peptides, the antiparallel arrangements can be favored by better backbone hydrogen bonding, backbone dipole−dipole interaction, and charge−charge interaction among the terminus residues. However, longer fragment such as Aβ1−28 favors antiparallel arrangement, while the parallel arrangement is favored by the short non-Aβ GNNQQNY sequence.170,181−184 Obviously, not only the length determines the stability of the fibril, but also the nature of the residues along the segment that interacts. For example, the available 15 microcrystal structures of cross-β spines exhibit parallel arrangements for peptides free of any charged residues.(43)
As can be seen from Table Table1,1, the percentage of the hydrophobic residues is above 40% for all β-strand segments presented here, and some of these segments are also enriched in hydrophilic residues. There are two hydrophobic rich regions in Aβ1−40/Aβ1−42 peptides, segments 17−22 (KLFFA) and 30−42 (AIIGLMVGGVVIA). ssNMR indicates that Aβ10−35, Aβ1−40, and Aβ1−42 fibrils are organized in parallel, whereas Aβ16−22, Aβ34−42, and Aβ14−23 are antiparallel. Most of these consist of hydrophobic segments believed to be crucial for aggregation.70,185
Hydrophobic interactions are the driving force for protein−protein association, including protein aggregation;(122) they largely control the stability and therefore determine the organization. In β-sheets, stabilization by hydrophobic interactions could maximize when the interactions are between stacked identical residues, which is the case in a parallel organization.(186) For example, 15 interactions between in-register hydrophobic residues of two β-strands stabilize the parallel organization of Aβ10−35, whereas there are only six interactions between hydrophobic residues in the antiparallel organization (Figure (Figure8A).8A). Parallel β-sheet organization juxtaposes the hydrophobic segments of neighboring peptide chains, while an antiparallel organization does not. Therefore, the preferred orientation of the β-strands for Aβ10−35 is parallel.
Illustration of the organization of the hydrophobic residues (black) and the charged residues (red), and their charge interactions (brown) in parallel and antiparallel alignment for (A) Aβ10−35, (B) Aβ16−22, and (C) Aβ ...
Aβ16−22 is stabilized by hydrophobic interactions between consecutive β-strands along the fibril axis. It seems that both parallel and antiparallel orientations are favored for Aβ16−22, with both arrangements presenting central hydrophobic sequence, including the strongest Phe−Phe hydrophobic interaction. However, Aβ16−22 has positively charged Lys at the N-terminus and negatively charged Glu at the C-terminus. Parallel β-sheet organization will create electrostatic repulsion destabilizing the fibril, whereas an antiparallel β-sheet organization simultaneously juxtaposes the central hydrophobic segment and minimizes the electrostatic energy (Figure (Figure8B).8B). Ma and Nussinov(83) studied the stability of Aβ16−22 and concluded that for the antiparallel model the hydrophobic interactions appear to dominate in this short segment. Nonetheless, as discussed in the next section, because hydrophobic interactions in the parallel Aβ16−22 organization are strong, they can overcome the electrostatic destabilization induced by flexible side-chains at the termini, leading to a parallel Aβ16−22 arrangement, as seen from Figure Figure8B.8B. Thus, while both organizations are likely to exist, the antiparallel one presents higher population. A similar picture is presented by Aβ34−42. Because it consists of a single hydrophobic segment with a positively charged N-terminus and negatively charged C-terminus, hydrophobic contacts maximize in either parallel or antiparallel structures, while electrostatic interactions would be expected to favor the antiparallel structure (Figure (Figure88C).
Hydrophobic interactions are nonspecific, which could underlie the diversity of possible interaction patterns. Therefore, amyloids dominated by hydrophobic interactions could easily present polymorphic behavior. In the case of Aβ16−22, an in-register antiparallel organization (Figure (Figure8BII)8BII) has been shown to be preferred. However, a new arrangement with one residue shifted and the backbone flipped (Figure (Figure8BIII)8BIII) is possible under acidic conditions.(82) It can be seen that both arrangements in Figure Figure8BII8BII and BIII present favorable hydrophobic interactions; however, the antiparallel one residue-shifted arrangement (Figure (Figure8BIII)8BIII) may have better aromatic interactions derived from two nearby Phe residues. The importance of aromatic interactions between two β-sheets in protein aggregation has been emphasized by Gazit and co-workers(187) who suggested that aromatic interactions play a key role in molecular recognition, self-assembly, and amyloid formation.(187) Makin and co-workers(188) further confirmed that aromatic residues that are capable of π−π stacking are important for amyloid fibril formation.
Apart from the role of the hydrophobic effect in the polymorphic character of the β-strands arrangement within β-sheets, the diversity of hydrophobic interactions between protofibrils can also contribute to amyloid polymorphism. In the case of Aβ1−40, the highly hydrophobic C-terminal region provides many possible interaction interfaces for 2-fold or 3-fold arrangement patterns (Figure (Figure66).73,138
Electrostatics can play a crucial role in Aβ fibrils.75,76,189,190 At first glance, the charged nature of many amyloid-forming peptides can increase the solubility and thus decrease their intrinsic ability to form β-sheet based aggregates. However, in certain cases, charged residues may help the peptides to form amyloids, as indicated by the full-length Aβ peptide, which forms ordered fibrils much more easily than the shorter and more hydrophobic aggregative Aβ17−42.
Table Table11 provides the net charge of the segments. Some Aβ segments have β-strands that are negatively charged, some are positively charged, and others have a net charge of zero. It appears that β-strand organization in parallel or antiparallel does not depend on the segment net charge. The percentage of the charged residues in the segment does not determine the arrangement of the β-strands either. However, charge−charge interactions between sheets are important for fibril formation and residues with complementary charges promote fibril formation. Therefore, the positions of the charged residues along the sequence of the segment are important for the stabilization of the amyloid fibril and affect the arrangement of the β-strands.
A parallel organization of Aβ forms homostacking (identical amino acids stacked on top of each other). Stacking of chemically similar charged residues in a parallel orientation creates electrostatic repulsion. In contrast, the antiparallel orientation is favored by electrostatic interactions between the C- and N- termini of neighboring molecules in a β-sheet (e.g., the positive amino and negative carboxylate groups at the ends of uncapped Aβ34−42 as illustrated in Figure Figure8)8) or by oppositely charge residues (e.g., positively charged Lys and negatively charged Glu side chains in capped Aβ16−22 as described in Figure Figure8).8). Thus, preferred fibril architecture maximizes the number of hydrophobic and attractive electrostatic interactions.
Because hydrophobic interactions are nonspecific, the attractive electrostatic interactions limit the variation of the amyloid arrangement. The Aβ11−25 fragment is a good example in this case. As compared to Aβ1−40, the Aβ11−25 fragment can form a fibril, which yields a better quality and higher resolution X-ray diffraction data (Figure (Figure33A),(77) indicating lower structural fibril diversity. Expanded from Aβ16−22, the charged pattern in Aβ11−25 increases the weight of electrostatic interactions as compared to the (unchanged) hydrophobic interactions (Figure (Figure3B).3B). In the N-terminus, two His residues (His13, His14) increase the positive charge in the Lys16 region, while Asp23 added to Glu22 leads to a more negative C-terminus. As a result, the strong electrostatic interactions make the in-register antiparallel alignment of Aβ11−25 highly favorable, with other alternatives hard to compete. Clearly, the strong electrostatic interaction contributed to the structural uniformity of the Aβ11−25 amyloid.
Preferred fibril architecture is also stabilized by tight van der Waals (vdW) packing between β-sheets. Steric zippers (that is, tight packing) appear a generic structural motif of the amyloid protofilament,170,191−193 because good geometrical fit provides favorable vdW interactions and constrains side-chain movement. For example, in the amyloid-like GNNQQNY fibril, the interactions are maximized by very tight side-chain packing.170,192 In the case of this all-polar sequence, the strong steric zipper match provides a dominant amyloid-like crystal, making an atomic resolution structure possible.
Studies of the stability of the islet amyloid polypeptide segments NFGAIL and NFGAILSS(194) have shown that interactions between hydrophobic residues and between aromatic residues dominate. However, recently other arrangements with good geometric fit between β-sheet layers have also been shown to be possible,(123) suggesting that for the islet amyloid polypeptide segment NFGAILSS, a tight steric zipper packing can occur in more than one way, increasing polymorphism. Recently, Park et al.(195) studied the polymorphism for the steric zipper patterns of various short peptides while considering the kinetics and the thermodynamics as driving forces for the structural selection.
For the Aβ11−25, a steric zipper can contribute to decreased polymorphism. The strong electrostatic interactions prefer the in-register antiparallel β-sheet. Considering only electrostatic interactions, the amyloid structure of Aβ11−25 can have an arrangement as in Figure Figure4H,4H, where electrostatic interactions are also maximized between sheets. Early work indeed suggested this architecture for the 11−24 region in Aβ1−28.(75) However, due to a better steric match between parallel β-sheets, the Aβ11−25 adopts the Figure Figure4G4G arrangement (also see Figure Figure3B)3B) to allow optimized hydrophobic, electrostatic, and steric zipper (vdW) fit.
The most prevalent metal ions in biological systems, such as Zn2+, Cu2+, and Fe3+, are known to be essential for normal brain function and development.196,197 Disrupted cellular homeostasis of these ions is thought to play a central role in the aggregation and neurotoxicity of Aβ.198,199 These metal ions are markedly enriched in Aβ plaques200−210 and may act as seeding factors. In contrast, while a higher concentration of Al3+ has been established in AD patients’ brain,211−214 the mechanism of its pathogenesis in AD is still debated.215−218 Nonetheless, experiments clearly demonstrated that Al3+ ions do play a role in Aβ aggregation and in neurotoxicity.219−224 Interactions with these metal ions may explain the increased involvement of soluble toxic oligomeric species.(225) To date, the conformation of Aβ complexes with metal ions is still a major challenge. Open questions include (1) which residues in the Aβ peptide interact with the metal ion; (2) are there different conformations with different residues coordinating the metal ions; and (3) among these, which are preferred. Characterization of the intrinsic conformational properties of Aβ1−40/Aβ1−42 oligomers complexed with metal ions is important to understand the effect of metal ions on Aβ aggregation. So far, Aβ binding sites for Zn2+ and Cu2+ were studied extensively, but there is lack of data for Fe3+.
Polymorphism can be observed in Cu2+ complexes with Aβ amyloid. Possible Cu2+ coordinating residues include histidines (His6, His13, His14), tyrosine (Try10), aspartate or glutamate (Asp1, Glu3, Asp7, Glu11, Glu22, Asp23), methionine (Met35), deprotonated amides of peptide backbone, or carbonyl groups.206,226−231 The most common residues are located at the N-terminus of Aβ: the binding site for Cu2+ was proposed to consist of the three histidines (His6, His13, and His14) and Tyr10.205,226,232−238 Further, studies demonstrated that the Cu2+ binding site can involve Asp1239,240 or Glu11.(240) A recent study of the Cu2+ binding site in the rat Aβ1−28 fragment revealed that Cu2+ binds to Asp1, His6, and His14.(241) His13 is absent in rat Aβ1−28. Recent simulations suggested that Cu2+ coordinates with His13 and His14 of each two Aβ peptides.(242) These studies suggest various possible conformers. We therefore conclude that Aβ amyloid with different experimentally detected Cu2+ binding sites depends on the populations of the various morphologies under different conditions.
The zinc binding site in Aβ is also located at the disordered N-terminal (Asp1−Lys16). Seven potential N-terminal residues can bind Zn2+: Asp1, Glu3, His6, Asp7, Glu11, His13, and His14. As for the Cu2+ binding site, in the case of Zn2+, the three histidines (His6, His13, and His14) at the N-terminal segment coordinate with Zn2+.226,227,237,243−260 Recently, three different models of Zn2+ binding to Aβ were observed by experiments: First, the NMR structures of Zn2+−Aβ1−16 in aqueous solution at pH 6.5 and 7.4 showed that Zn2+ is bound to these three histidines and to the two carboxylic groups of Glu11.(252) Second, an NMR study on Zn2+−Aβ1−28 in sodium dodecyl sulfate (SDS) micelles at pH 7.5 and a temperature of 298 K proposed a tetra-coordination of Zn2+ by His6, Glu11, His14, and Asp1 at the N-terminus for rat Aβ1−28, and penta-coordination of Zn2+ by His6, Glu11, His13, His14, and Asp1 at the N-terminus for human Aβ1−28.(257) The third model is based on X-ray absorption spectroscopy (XAS) in which Zn2+−Aβ complexes are coordinated with four histidines (His13 and His14 of two monomers), indicating that each metal ion is shared by two Aβ peptides.(258) Aβ oligomers coordinated with Zn2+ can be modeled using experimental data as shown in Figure Figure9.9. See an additional recent reference in the note added in proof. We therefore propose that Zn2+ can coordinate the N-terminal domain in different Aβ1−42 oligomer morphologies. The prevailing organization and the probability to detect certain morphologies depend on experimental conditions such as temperature, Zn2+ concentration, and pH. Oligomer stability depends also on its size, in particular when dealing with Zn2+ in the N-terminal.
Much less is known about the preferential regions of Al3+ binding to Aβ. Fasman(261) proposed that Al3+ coordinates with Asp, Ser, Tyr, and Glu in Aβ. Recently, Ricchelli et al.(262) suggested that Al3+ may coordinate with residues in both the hydrophilic segment (1−16) and the hydrophobic sequence 20−35 in the Aβ peptide, and thus Al3+ binding site is less restricted than that of Zn2+ or Cu2+, which are confined to the N-terminal. Thus, polymorphic Al3+ Aβ complexes are also expected.
Amyloid toxicity is the outcome of Ca2+ conductance through the membrane, and, as described below, polymorphic Aβ oligomers can differ in their mechanisms of toxicity. Aβ toxicity was first identified by formation of Aβ1−40 cation selective channels in planar lipid bilayers fused with liposomes and in acidic bilayers.(263) The Aβ1−40 channel in the solvent-free membrane patch presented multiple cation selectivity and sensitivity to tromethamine, which suggested that in cells the ion fluxes would disrupt cellular homeostasis.(263) Toxic Ca2+ conductance through unregulated pores or ion channels formed by Aβ or its fragments was also supported by other studies264−267 and appears universal for amyloidogenic peptides. The channels are polymorphic48,50 and differ from conventional ion channels.(268) Even though it is currently believed that small oligomers play critical roles in ion-channel formation, the relationship between amyloid deposition and cellular toxicity is still controversial, and the similarity to bacterial pore-forming toxins is challenging to study.(269)
Other soluble Aβ forms, which are broadly named as amyloid β-derived diffusible ligand (ADDLs), have different morphologies and were proposed to have different mechanisms. Unlike ion channels observed in bilayers, ADDLs were shown to be linked to neuron toxicity, mostly via binding to protein receptors.(270) ADDLs are highly ordered, binding to a specific subset of postsynaptic proteins. In particular neural signal transduction pathways, ADDLs can impair synaptic plasticity and associated memory dysfunction during the early stage of AD.
In 2005,(271) it was observed that a homogeneous and stable globular amyloid β-peptide oligomer can be easily prepared in vitro. This globular amyloid β-peptide was present in the brains of patients with AD and in Aβ1−42-overproducing transgenic mice. The pure, water-soluble globular 60 kDa oligomer was reported to have 12 Aβ monomers. This “globulomer” is a persistent structural entity apparently formed independently of the fibril aggregation pathway. Unlike ADDLs, the Aβ1−42 globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Similar to ADDLs, the globulomer also relates to early AD. In 2006, another species smaller than the globular 60 kDa oligomer, Aβ*56, was reported as a soluble oligomer causing memory deficiency in middle-aged mice.(225) However, on the basis of biochemical properties and immunospecificity, it was argued to be unlikely to bind to receptor proteins, and instead to impair memory independently of plaques or neuronal loss.(225) A larger soluble oligomer, “amylospheroids” (ASPDs),272,273 was also observed, with sizes around 10−15 nm. These spherical Aβ assemblies have high-mass (more than 100 kDa) and can be detected in AD and Lewy body (DLB) brains, using ASPD tertiary structure-dependent antibodies. ASPDs bind presynaptic targets on mature neurons with different toxicity from other ADDLs. Finally, cellular prion proteins (PrPCs) were recently identified as high-affinity cell-surface receptor for soluble Aβ oligomers on neurons,(274) but the interaction does not require infectious PrPSc conformation.
Even though many studies argue that soluble Aβ oligomers such as ADDLs are off fibril formation pathways, they may nonetheless share some structural similarity with protofibrils. An NMR study of the intermediates of globulomer formation, the so-called preglobulomer, suggested parallel in register β-sheets in the C-terminal region, even though the N-terminal region has different conformation.(275)
Overall, the observations described above lead to the conclusion that the inherent polymorphic nature of Aβ assemblies can be associated with different toxic behavior. Consequently, there is a need to delineate the structural variability underlying the formation and stabilities of soluble Aβ oligomers.
The native-like states of amyloidogenic proteins are unstable in environments favoring aggregation, leading to a broad conformational heterogeneity. Among the native-like states, some display amyloid-favored populations. While these conformations may have higher-energy and consequently exist at low concentrations, they may be complementary to a fibrilar state surface. These binding-favored conformers would associate via the “conformational selection” mechanism. Binding stabilizes these conformations, and shifts the population of the monomers toward these binding-favored conformers, propagating the binding reaction.57,58,176,177,276−280 The larger are the aggregates, the more stable are the associations (Figure (Figure10).10). Amyloid formation follows the conformational selection and population shift mechanism now accepted as a general paradigm in molecular recognition.(56) This view is based on a dynamic energy landscape picture of protein folding and protein binding. The kinetics of the amyloid formation process depends on the relative stabilities of the states, and the barrier heights that need to be crossed in the conformational transitions, which are the outcome of environmental conditions.
Polymerization of Aβ peptides involves continuous hierarchical redistributions of the polymorphic ensembles on a rugged energy landscape. For illustration, we present five aggregation phases starting from the native states and ending in the fibril ...
Protein conformations and their associations have been optimized by evolution toward specific biological functions. Thus, it is expected that certain states are favored over others due to (some) energy gaps between the favored and the less-favored states; this, however, is not the case in amyloid formation, which is a misfolded, diseased state presenting a rugged energy landscape. Because the toxic non-native amyloidogenic conformations are not under selection pressure to preferentially assemble into a specific oligomer, the outcome is an extensive polymorphic range. As the heterogeneous seeds and fibrils grow, the ordered polymorphic aggregates will increasingly stabilize. Metal ions, such as Zn2+, Cu2+, and Fe3+, known to promote amyloid formation, further complicate these scenarios. Each Aβ conformer can coordinate metal ions via different residues; because the differences in the stabilities may be not large, the complexity of the landscape, which reflects the polymorphic range, vastly increases.
The nature of the rugged energy landscape of amyloidogenic peptides provides a conceptual framework for polymorphic stages in protein aggregation. As shown in Figure Figure10,10, we may describe five protein aggregation phases, with hierarchical redistribution of the ensembles. The “normal” folded stage (Figure (Figure10A)10A) and partially unfolded or aggregated state (Figure (Figure10B)10B) have been universally regarded as necessary steps. However, where to position the “soluble oligomers” with respect to the main fibril forming pathway is still unclear. Even though small amyloidogenic oligomers are on the pathway to seed and larger protofibril formation, ADDL-like species were suggested to be off pathway (for example, as depicted by Roychaudhuri et al.(281)). Such view overlooks common features of ADDLs and other β-sheet-rich polymer forms.(275) Here, we call attention to the possible existence of certain “critical intermediates” (Figure (Figure10C),10C), which can lead to both seeds and other soluble ADDL-like oligomers (Figure (Figure10D).10D). From this point of view, all soluble oligomers are polymorphic forms distributed on the energy landscape prior to (proto-)fibril formation (Figure (Figure10E).10E). Thus, in principle, a drug can inhibit such critical intermediates, preventing seed and other ADDL-like oligomer formation.
Conformational selection and population shift is the key mechanism in biomolecular recognition,(56) and adding monomers and small oligomers to amyloid seeds in fibril growth(177) is a molecular recognition event.(54) Within such general framework, the “dock and lock” mechanism for the deposition of soluble Aβ onto amyloid was proposed to be mediated by two distinct kinetic processes. In the first “dock” phase, the addition of Aβ to the amyloid seeds can be reversible, while in the second “lock” phase, the deposited peptide becomes irreversibly associated with the template in a time-dependent manner.(282) In particular, in 2001 Massi and Straub combined the “dock−lock” mechanism with the energy landscape to explain the Aβ amyloid growth,(283) thus unifying a number of schemes proposed to describe fibril elongation, is in good agreement with experimental observations for Aβ amyloid(283) and prion growth.(284)
Extensive molecular dynamics simulations of Aβ16−22 in water(88) and urea(285) indicated that both hydrophobic interactions and backbone hydrogen bonds are important for Aβ16−22 oligomer stability. The kinetics of dissociation of the Aβ16−22 oligomer(286) and oligomer growth(287) both involve dock and lock stages. Importantly, it was found that the mobile structured oligomers undergo large conformational changes to accommodate the added monomer, a clear indication of mutual conformation selection process.(287)
Simulation of fibril growth of other fragments, Aβ35−40 (MVGGVV),(288) Aβ10−40,(289) and Aβ10−40 with an Asp23Tyr mutation,(290) pointed to a two-stage mechanism.282,283 The simulations of Aβ10−40 emphasize that the docking transition appears to be continuous and it occurs without free energy barriers or intermediates. Still, it should be distinguished here between short peptides and proteins, short peptides being random coil in solution. During the docking transition, incoming Aβ monomers are disordered on the fibril edge. The locking stage is characterized by the rugged free energy landscape. The locking step takes place when an incoming Aβ peptide forms a parallel β-sheet structure on the fibril edge. Because the β-sheets formed by locked Aβ peptides are typically off-register, the structure of the locked phase differs from the structure of the fibril interior.(289) Polymorphic forms can also derive from a variation of pre-existing seeds. Finally, docking291−293 and the role of conformational ensembles in docking associations(56) are general paradigm in protein and biomolecular recognition.
Experimental and computational studies of Aβ amyloids suggest that for any given segment there are one or more preferred parallel and (or) antiparallel structural states. The preferred organizations of the Aβ fragments do not appear to present straightforward “rules” with respect to length, hydrophobicity, charge, etc. Because polymorphism is presented by different Aβ segments, clearly a combination of these segments would lead to polymorphic full-length Aβ, although the relative populations in the full sequence are likely to be different. This leads us to argue that the observed polymorphism for the disease-related species derives from combinations of the preferred fragment architectures. Because these are strongly affected by the environment, the trade-off among these fragment-interactions and the key nucleation sites may determine the polymorphic oligomer (and fibril) outcome, particularly with respect to parallel/antiparallel backbone alignments.
How the Aβ peptides assemble and form toxic entities and what is the mechanism of toxicity are major questions that persist in Alzheimer research. To address such questions, it is essential to have working structural models of small Aβ oligomers. Two types of models of the three-dimensional structures of Aβ oligomers have been reported from computational and experimental studies. The first is the Ma−Nussinov−Tycko model,46,68 and the second is by Lührs et al.,(15) indicating polymorphism derived from different turn structures. Additional amyloid organizational motifs containing a U-turn have been reported.150,191,192 Architectures for the same sequence, which include such a U-turn in their structure, and have similar parallel organization, may be different, due to different turn conformations. The resulting side-chain packing can affect the left-handed twist along the main axis of the amyloid fibril, leading to different fibril morphologies. Nonetheless, it is important to bear in mind that these may only present the prevailing organizations under the experimental conditions; subpopulations with different morphologies may not be captured. The further association of the oligomeric forms leads to combinatorial increase of polymorphic populations at the supramolecular and matured fibril levels. Polymorphism can also be demonstrated by metal ion binding to Aβ oligomers. Because metal ions can coordinate with different residues in each structural model, the variety of the morphologies can increase dramatically.
It behooves us to emphasize, however, that here we review polymorphism focusing on larger scale fibril organization. We do not discuss polymorphism as relating to small side-chain conformational variability and its effects on the aggregated states. This is because current experimental data, based on ssNMR, cryo-EM, fiber diffraction, and mutational studies, are unable to provide such high resolution detail. We further only touched on the effects of pathways and intermediate states because experimental data are scant. Different pathways can lead to different preferred conformational states; however, pathways are strongly dependent on conditions, for example, concentration, sequence effects, pH, and temperature.121,294−298 Our focus on Aβ-derived peptides may miss structural contributions like hydrogen-bond ladders, as, for example, for in-parallel stacked Asn or Gln, which also greatly enhances the stability of a parallel orientation.(186)
Nevertheless, the Aβ-derived peptides provide an excellent example toward the understanding of amyloid polymorphism. While here we highlighted hydrophobic interactions as contributing to an increase in polymorphic amyloid behavior, electrostatic interactions could decrease the variability, and steric zipper (vdW) effects may increase or decrease polymorphism depending on the geometric match. Eventually, for amyloid polymorphism, both dominant and minor forms should all be treated in addressing the mechanism of aggregation and toxicity, because it is always possible that minor species are the toxic agents. Therefore, comprehension of polymorphism should assist in therapeutic agents design.
Currently, it is increasingly recognized that amyloids present a vast range of conformational states. Their energy landscape is rugged. Complementary protein conformations are selected from equilibrium of low-energy and higher-energy conformations. This “selected-fit”,(280) or conformational selection mechanism,57,58,176,177,276−279 is based on a dynamic energy-landscape picture of protein folding. The polymorphic differences may or may not be large; polymorphism can be expressed in altered β-strands arrangements, altered turn conformations, altered sheet−sheet registrations, differences in the locations and fragment sizes of regions that are disordered, different sheet organizations, and different supramolecular packing. Because these have not been optimized by evolution for a particular function, the differences in stabilities between the states are likely to be small, with the distribution of the populations easily shifting under even slight changes in cellular conditions. Because amyloids are involved in human disease, comprehension of the range of (preferred) states on their own and when interacting with metal ions is vital. Here, we addressed this challenging problem, reviewing available data within this framework, focusing on the Aβ.
Polymorphism raises a key question: how to prevent AD with drug design or other therapeutic approaches. Targeting toxicity in late stage AD may miss the reservoir of polymorphic forms; preventing Aβ production may encounter side effects derived from normal Aβ function loss. If common links among polymorphic forms can be established, then targeting the bottleneck would be more effective. The “critical intermediates” (for example, as in Figure Figure10C)10C) could be the main AD targets.
We thank current and former group members Drs. Hyunbum Jang, Jie Zheng, and David Zanuy for their computational studies cited here and Drs. Ratnesh Lal and Bruce Kagan for many discussions. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract number HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Yifat Miller received her Ph.D. degree in Computational Physical Chemistry from the Hebrew University of Jerusalem in 2007 under the supervision of Professor R. Benny Gerber, using molecular dynamics simulations to investigate the chemistry of atmospheric molecules. She is currently a NIH postdoctoral fellow in the laboratory of Professor Ruth Nussinov at the CCR Nanobiology Program, National Cancer Institute. Her current research interest focuses on the structural variability of potential conformations of Alzheimer’s Aβ under various conditions and the self-assembly mechanism leading to ordered fibril formation.
Buyong Ma is a senior scientist at SAIC-Frederick, Inc., National Cancer Institute at Frederick. He received his B.Eng. degree in polymer chemistry from the Hefei University of Technology in 1984. He received his Ph.D. degree in physical chemistry from the University of Georgia at Athens in 1995. From 1995 to 1998, he was a postdoctoral researcher with Professor N. L. Allinger, focusing on development and application of molecular mechanics. In 1998, he joined the National Cancer Institute (NCI) as a Research Fellow and started his research in computational biology. He was a research scientist in Locus Pharmaceuticals from 2002 to 2003. In 2003, he returned to NCI as a senior scientist at SAIC-Frederick, Inc. He has authored over 100 scientific papers. His research interests and philosophy are to use computational approaches as “experimental tools” to study protein−protein interaction, protein−nucleic acid interaction, and protein aggregation, at molecular level and systems biology level.
Ruth Nussinov received her Ph.D. in 1977, from the Biochemistry Department at Rutgers University, and did postdoctoral work in the Structural Chemistry Department of the Weizmann Institute. Subsequently she was at the Chemistry Department at Berkeley, the Biochemistry Department at Harvard, and the NIH. In 1984, she joined Tel Aviv University. In 1990, she became a Professor in the Department of Human Genetics, at the Medical School. In 1985, she accepted a concurrent position at the National Cancer Institute of the NIH, where she is a Senior Principal Investigator heading the Computational Structural Biology Group. She has authored over 350 scientific papers. Her interests largely focus on protein folding, protein−protein interactions, amyloid conformations, and large multimolecular associations with the goal of understanding the protein structure−function relationship.
National Institutes of Health, United States
Since the completion of this review, the polymorphic states of Aβ oligomers coordinated with Zn2+, using the experimental data cited in this review in Section 6 and in Figure Figure9,9, were described in Miller et al. See ref. 299.