Rodriguez JA, Ivanova MI, Sawaya MR, Cascio D, Reyes FE, Shi D, Sangwan S, Guenther EL, Johnson LM, Zhang M, Jiang L, Arbing MA, Nannenga BL, Hattne J, Whitelegge J, Brewster AS, Messerschmidt M, Boutet S, Sauter NK, Gonen T, Eisenberg DS. Structure of the toxic core of α-synuclein from invisible crystals. Nature. 2015 Sep 24;525(7570):486-90. Epub 2015 Sep 9 PubMed.

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  1. This highly interesting study by Eisenberg and colleagues reveals the atomic resolution structure of two short segments of α-synuclein in a fibril-like state. Using micro-electron diffraction. the structure of an 11-residue segment of α-synuclein, which is at the core of amyloid fibrils of full-length α-synuclein, has been resolved in nanocrystals down to a fascinating resolution of 1.4 Å. Together with the structure of another short segment containing the site of a genetic mutation (A53T), an interesting model for an approximate 30-residue fragment of α-synuclein in amyloid fibrils was proposed.

    The proposed model goes beyond what has been previously possible on the basis of X-ray crystal structures of other short peptides, as it includes an experimentally supported turn at Gly51. The turn is important as high-resolution solid-state NMR studies of Aβ peptides have shown that amyloid fibrils of longer polypeptides are not only composed of near ideal, stacked β-sheets, but can have a variety of kinks and turns. The positioning of the NACcore at the center of the proposed model is in agreement with previous NMR studies (e.g., our work as in Cho et al., 2011), which showed that in amyloid fibrils of full-length α-synuclein the NACcore segment is most protected from hydrogen-deuterium exchange. At present, it remains open how the additional β-strands—according to solid-state NMR most polymorphs of full-length α-synuclein characterized so far contain at least four to five β strands—will be arranged in amyloid fibrils of full-length α-synuclein.

    In the first solid-state NMR study of α-synuclein fibrils performed by the Baldus group (Heise et al., 2005) different polymorphs of amyloid fibrils of full-length α-synuclein were reported, and several more have been described in recent years. The high-resolution structure of the two short segments of α-synuclein in nanocrystals, as revealed now by micro-electron diffraction, does not yet provide direct information about potential differences in the structure of different polymorphs of α-synuclein fibrils. However, in the two strains of α-synuclein recently reported (Bousset et al., 2013; Peelaerts et al.,  2015), the NACcore region appears to be in a β-strand conformation, such that in both these strains the model proposed by Eisenberg and colleagues might represent the core of the amyloid fibrils.

    The two short segments for which the high-resolution structure of an aggregated state has now been solved by Eisenberg and colleagues are clearly two regions that are highly important for the neurotoxicity of α-synuclein. Further studies are now required to find out how well these two segments recapitulate the neurotoxic properties of full-length α-synuclein. To this end a variety of aspects should be considered. This includes differences in toxic effects such as membrane permeabilization on the one hand, and the ability to lead to efficient spreading of pathology on the other. For example, amyloid fibrils might be more efficient agents for spreading of pathology (Taschenberger et al., 2011), while intermediate aggregation states such as soluble oligomers are likely to most strongly perturb cellular membranes.

    In summary, the study by Eisenberg and colleagues is an important step toward the high-resolution structure of amyloid fibrils of α-synuclein taken from different strains.

    View all comments by Markus Zweckstetter

  2. Rodriquez and colleagues provide the first high-resolution structure illustrating how regions of individual α-synuclein monomers contact other monomers in order to form the β-sheet-rich amyloid fibrils that are found in the characteristic Lewy bodies and neurites that are the hallmark of Parkinson’s disease and other synucleinopathies. Notably, the authors characterize the structure of microscopic crystals formed by an 11-residue segment of the NAC domain of α-synuclein, which has long been considered to contain the key elements required for synuclein aggregation (for example, by Giasson et al. and by Bodles et al.). This segment of the protein contains sequence elements that differentiate α-synuclein from its family members β- and γ-synuclein, and has been pinpointed previously.

    In the crystal structure, α-synuclein residues 68-78 form a single β strand. Such a long β strand has not been previously observed in crystal structures of other amyloidogenic peptide fragments, and is posited by the authors to be responsible for the microscopic nature of the crystals that they obtained. The formation of a β strand by this particular region of α-synuclein within fibrils has been consistently documented in a number of previous solid-state NMR studies, including those from the groups of Marc Baldus, Roland Riek, Chad Rienstra, and Beat Meier, though the presence of glycine 73 in the middle of this segment has raised some questions regarding the possibility of a kink or interruption of the β-strand structure.

    The crystal structure also reveals how individual copies of this segment interact with other copies to form the spine of the resulting amyloid fibrils. These highly specific interactions have proven difficult to delineate using solid-state NMR or other techniques for α-synuclein, although considerable advances have been made for other amyloids by the groups of, among others, Rob Tycko, Beat Meier, and Robert Griffin. Nevertheless, the reported structure is that of a typical twofold symmetric steric zipper, formed by two individual strands, in the characteristic manner that has now been observed by the Eisenberg group for many amyloidogenic peptides (although the presence of two water molecules within the interface is noted as an anomaly, since amyloid steric zippers typically exclude water completely). The biggest surprise and advance contained in the present work is the identification of a second interaction interface for amino acids 68-78, formed on the opposite side, i.e., the “outside” of the steric zipper, which is presumed to define the innermost core of the fibrils. Furthermore, a second synuclein fragment, consisting of residues 47-56, is found to form amyloid fibrils with an atomic interface that almost exactly mimics that formed on the “outside” of the 68-78 peptide, and features nearly identical amino acid sequences. The authors draw the obvious conclusion, namely that 47-56 of α-synuclein may fold back to pack onto the outside of the innermost fibril core structure formed by residues 68-78. This enables them to build a highly detailed model for the amyloid fibril of α-synuclein, in which the innermost core interface and the packing of a secondary layer are delineated at atomic resolution.

    The new insights offered by this work have several implications for future research. First, the authors note that the A53T mutation, which was used in their constructs, likely enhances packing at the secondary interface, perhaps explaining why this mutation aggregates more efficiently and is associated with disease. It will certainly be interesting to determine and/or model the effects on this interface of the other known PD mutations in this region, H50Q, G51D and A53E. In addition, the current work does not inform about the conformation that other parts of α-synuclein may adopt in the fibril structure. On the C-terminal side, residues up to position 95 are known to be ordered within α-synuclein fibrils, and on the N-terminal side, some fibril forms exhibit order until the very N-terminus, though other forms only extend to residue 38 or so.  This leaves a large portion of the fibril structure that needs to be determined and reconciled with the current model. Furthermore, the region between the 47-56 segment and the 68-78 segment is also found to adopt β-strand structure in at least some fibril forms, suggesting that this region may play a more important role than simply that of a connecting loop.

    Intriguingly, recent progress in the study of several amyloids has suggested that different disease strains may correspond to different structural strains of the associated amyloid fibrils, and this hypothesis has been extended to synuclein fibrils in recent work from the Lee/Trojanowski and Melki/Baekelandt groups. Certainly, a number of morphologically distinct α-synuclein fibril forms have been characterized by different groups, and it is clear that at least some details of their molecular structures differ. It will be interesting to see whether the micro-electron diffraction technique used to obtain the current structures will be able to provide information on fibrils formed by additional α-synuclein segments and constructs, and whether such additional structures may offer deeper insights into the links between amyloid structure and disease etiology and presentation.

    View all comments by David Eliezer

  3. In this study, Schmidt and colleagues elucidate the Aβ42 dimer structure within the Aβ42 fibril by cryo-electron microscopy (cryo-EM). This is a follow-up from the previous study of the same group (Schmidt et al., 2009), in which cryo-EM was applied to examine possible models of Aβ40 and Aβ42 fibrillar structures that might be consistent with the experimental constraints of the technique.

    Interestingly, in this earlier study two protofilaments of Aβ40 fibrillar structure were identified (one associated with a more structured N-terminal region than the other), whereas a single protofilament characterized Aβ42 fibrillar structure. The present study focuses on the Aβ42 fibrillar structure (Schmidt et al, 2015). The Aβ42 dimer unit of the protofilament consists of the central region containing two oppositely oriented C-terminal segments (each 23-26 amino acids long) forming a β-sheet structure within the parallel cross-β fibril, while the two peripheral regions formed by the two N-terminal segments (the first 10-12 amino acids in the sequence) flank the central region but are significantly less structured than it. This overall structure, based on an Aβ42 dimer unit within the fibril, differs from the Aβ42 fibril structure reported earlier this year by Ishii and collaborators, who used solid-state NMR (Xiao et al., 2015). The most obvious difference between the two is the Aβ42 tertiary fold in the decapeptide region Aβ42 (21-30) detected by the solid-state NMR, which appears to be absent from the cryo-EM fibrillar structure. This U-shaped turn structure characterizes the Aβ40 fibril model proposed by Rob Tycko and collaborators and its absence from the cryo-EM-derived Aβ42 fibrillar model is a novel and unexpected feature. Notably, the extended central region of the cryo-EM Aβ42 fibrillar structure is larger than the corresponding region of the cryo-EM Aβ40 fibrillar structure, which explains the observation that Aβ40 and Aβ42 fibrils do not mix well in vitro.

    The observations of Schmidt et al. are consistent with oligomer structure predictions of our computational study (Urbanc et al., 2004), which showed that (a) the C-terminal region of Aβ42 monomer and oligomer conformations is significantly more structured than the C-terminal region of the corresponding Aβ40 conformations; (b) Aβ42 oligomerization is dominated by interactions between C-termini, whereas the central hydrophobic cluster plays the dominant role in Aβ40 oligomerization; and, most importantly, the N-termini of Aβ42 oligomers are significantly more flexible and disordered than the N-termini of Aβ40 oligomers, a feature that was hypothesized to mediate toxicity of Aβ42 oligomers (Urbanc et al., 2011; Dec 2010 conference news). Schmidt et al. posit that Aβ42 fibril is formed through self-association of dimers and/or their multiples, which would imply that some structural features of oligomers may be preserved in the fibril structure, which appears to be in agreement with computational studies. Moreover, Aβ42 fibril growth through dimer/multimer association is also consistent with the general biophysical principles of the minimal self-assembly model, recently reported by my group (Barz and Urbanc, 2014), which predicts a nucleated, structural conversion from disordered quasi-spherical to more ordered, elongated, protofibril-like structures. 

    References:

    Schmidt M, Sachse C, Richter W, Xu C, Fändrich M, Grigorieff N. Comparison of Alzheimer Abeta(1-40) and Abeta(1-42) amyloid fibrils reveals similar protofilament structures. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19813-8. PubMed.

    Xiao Y, Ma B, McElheny D, Parthasarathy S, Long F, Hoshi M, Nussinov R, Ishii Y. Aβ(1-42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease. Nat Struct Mol Biol. 2015 Jun;22(6):499-505. Epub 2015 May 4 PubMed.

    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. PubMed.

    Urbanc B, Betnel M, Cruz L, Li H, Fradinger EA, Monien BH, Bitan G. Structural basis for Aβ1–42 toxicity inhibition by Aβ C-terminal fragments: discrete molecular dynamics study. J Mol Biol. 2011 Jul 8;410(2):316-28. PubMed.

    Barz B, Urbanc B. Minimal model of self-assembly: emergence of diversity and complexity. J Phys Chem B. 2014 Apr 10;118(14):3761-70. Epub 2014 Mar 6 PubMed.

    View all comments by Brigita Urbanc

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