An ultra-high-resolution structure of the core segment of assembled α-synuclein — the protein that aggregates in the brains of patients with Parkinson's disease — has been determined. A neurobiologist and a structural biologist discuss the implications of this advance. See Article p.486
The paper in brief
- A small segment of α-synuclein is thought to form the core of the protein fibrils that are associated with Parkinson's disease and other synucleinopathies.
- On page 486 of this issue, Rodriguez et al.1 used the sophisticated electron-diffraction technique MicroED to determine the structure of this tiny core segment of just 11 amino-acid residues.
- The 1.4-ångström structure is the highest resolution yet achieved through cryo-electron-microscopy methods.
- The authors also present a MicroED structure of a segment of assembled α-synuclein that is mutated in some cases of familial Parkinson's disease.
The abnormal assembly of α-synuclein is central to Parkinson's disease2. Fibrils formed from α-synuclein (Lewy pathology) are seen in some brain neurons of more than 95% of patients with the disease, and their formation is associated with neurodegeneration3. Certain mutations in the α-synuclein gene, SNCA, and multiplications thereof, cause rare cases of Parkinson's disease. Sequence variants in the gene's regulatory region are associated with increased disease risk. Moreover, overexpression of mutant human α-synuclein in animal models causes its aggregation and neurodegeneration.
α-Synuclein is a 140-amino-acid protein that is abundant in nerve cells, where it is concentrated in nerve terminals. The protein binds to lipids through its amino-terminal half, which comprises seven imperfect repeat sequences. Upon lipid binding, α-synuclein takes on a partly α-helical structure. Under pathological conditions, it self-assembles into oligomers and fibrils. A seed of assembled α-synuclein can trigger aggregation of the soluble protein, and these insoluble aggregates slowly propagate through the brain. The long interval between the formation of the first protein inclusions and the appearance of disease symptoms opens a therapeutic window, provided that sufficiently sensitive diagnostic techniques can be developed.
Unbranched α-synuclein fibrils are 5–10 nanometres in diameter and up to several micrometres long. They assemble from the full-length protein, but only approximately amino acids 30–100 make up the structured part. Mutations known to cause Parkinson's disease are located between residues 30 and 53. Like other insoluble fibrous protein aggregates (known as amyloids), α-synuclein fibrils contain linked β-sheet structures.
Rodriguez and colleagues present structures of an 11-amino-acid peptide corresponding to residues 68–78 of α-synuclein (Fig. 1), and of a peptide of residues 47–56 that contains the disease-causing mutation A53T. What evidence is there that residues 68–78 form the core of α-synuclein fibrils? Previous work has shown that deletion of residues 71–82 abolishes the ability of α-synuclein to assemble into fibrils, propagate and be neurotoxic, and that a peptide of these amino acids will assemble into fibrils. Similarly, deletion of residues 66–74 abolishes assembly and this peptide can also form fibrils. Residues 68–78, studied by Rodriguez et al., can assemble into fibrils too4, and the electron-diffraction pattern produced by these assemblies resembles that of fibrils made from full-length α-synuclein. The authors show that fibrils of peptide 68–78 are toxic when externally applied to cells from tumour lines. However, aggregates of α-synuclein form inside cells in Parkinson's disease, so the relevance of this extracellular toxicity is not clear.
Previous studies of fibrils assembled from full-length α-synuclein have shown that residues 68–78 make up one of several β-strands. Although a complete description of the fibril will require the atomic structures of all β-strands and the regions in between, Rodriguez and colleagues' findings echo previous atomic structures of amyloid-forming peptides5. The structures show paired β-sheets with parallel β-strands in each sheet and antiparallel β-strands between sheets (Fig. 1). However, the zipper structure that marks the region between the paired sheets is longer than in other structures, and each pair of β-sheets contains two water molecules, instead of being dry. This new structural information may contribute to the development of molecules that can inhibit the formation of α-synuclein fibrils, as has been shown for the aggregates of tau proteins associated with Alzheimer's disease6.
“The authors postulate that this mutated region interacts with the fibril-forming core to enhance aggregation.”
The structure of the assembled mutated A53T α-synuclein peptide shows pairs of interdigitating β-sheets, but with β-strands kinked at residue 51. The authors postulate that this mutated region interacts with the fibril-forming core to enhance aggregation. It remains to be seen whether this model can account for the effects of other mutations in this region that cause Parkinson's disease. Furthermore, the fact that the mutations A30P and E46K, which also cause Parkinson's disease, lie outside the regions studied, suggests that further structural surprises may be in store.
The toxic cores of α-synuclein form well-ordered three-dimensional crystals (Fig. 1). But these crystals are so small that they are invisible by light microscopy, and are thus not amenable to structure determination using conventional X-ray crystallographic techniques, or even X-ray free-electron laser (XFEL) technology. Now, Rodriguez et al. have solved the crystals' structure using MicroED — a method based on cryo-electron microscopy (cryo-EM).
Cryo-EM encompasses several techniques used in structural biology. Single-particle cryo-EM, which determines structures by averaging images of many individual molecules, has already produced high-resolution structures of molecules that were for decades beyond the reach of other crystallographic methods7. Electron crystallography has also produced atomic structures from molecules that form 2D crystals. The development of MicroED, which uses electron diffraction to determine the structure of microscopic 3D crystals8, has added a new method to the cryo-EM repertoire.
Electron diffraction is an established EM technique that has been used to determine the atomic structures of membrane proteins, such as water channels, that form well-ordered 2D crystals9. But for 3D crystals, the situation is more complicated. The primary concerns are dynamic (multiple) scattering, and an inability to index and merge diffraction patterns collected from crystals that vary in size and morphology. MicroED resolves the indexing problem by tilting the crystal in the electron microscope and collecting multiple diffraction patterns from a single crystal, in much the same way as in X-ray crystallography. Continuous rotation of the crystals during data collection also attenuates the dynamic-scattering effect10.
“The structures are the first to be determined by MicroED from a molecule of previously unknown structure.”
Rodriguez and colleagues' structures are the first to be determined by MicroED from a molecule of previously unknown structure. They are also the highest-resolution structures determined using any cryo-EM technique. The study thus demonstrates the tremendous potential of MicroED for use in cases where other crystallographic methods cannot be used. The interaction between an electron beam and a specimen is much stronger than with X-rays, enabling the collection of high-quality diffraction data from tiny crystals. As predicted more than 15 years ago11, the charge of an electron makes it relatively easy for electron diffraction to visualize charged atoms in a structure, such as protons, which require high resolution to be resolved by X-ray crystallography. The instrumentation is readily available, in that an electron microscope can be operated either as a microscope to produce an image of the specimen, such as for single-particle cryo-EM, or as a diffractometer to produce a diffraction pattern of the specimen. The data-collection and processing methods involved in MicroED are similar to those used in X-ray crystallography and, in comparison with XFELs, the instrumentation cost is amazingly low and accessibility substantially greater.
MicroED does have its limitations. The 'phase problem' of crystallography — the fact that the phases of diffractions cannot be measured — can be particularly challenging in this approach. It is not easy to change the wavelength of an electron beam, so the technique of using multi-wavelength anomalous diffraction to determine phase will probably not be applicable. And it is not clear whether isomorphous replacement, in which heavy metal atoms are inserted into the structure, will work for ab initio phasing, because dynamic scattering may reduce the diffraction signals generated by heavy metals. So far, all structures determined by MicroED used the molecular-replacement method for phase determination, but it remains to be seen how the phasing problem will be resolved in future studies.
There is probably also a constraint on the size of crystals that can be studied by MicroED. The strong scattering makes large crystals impenetrable by electron beams, and merging diffraction patterns from different crystals may be difficult with crystals that have only a few unit cells in one direction. Nonetheless, MicroED provides another highly promising and complementary tool for structural biologists.
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