Protein aggregates associated with neurodegenerative disease have stubbornly resisted researchers’ efforts to get a good look at them. They refuse to crystallize well or yield to standard spectroscopic techniques. Now, advances in electron microscopy methods are forcing these molecules to give up their secrets. In the September 9 Nature, researchers led by David Eisenberg at the University of California, Los Angeles, and Tamir Gonen at the Howard Hughes Medical Institute’s Janelia Research Campus in Ashburn, Virginia, offer the closest look yet at the core of α-synuclein aggregates. The researchers made microscopic crystals from the peptides and used a relatively new technique called micro-electron diffraction to map them down to atomic resolution. Commenters called the work a tour de force.
“[These] structures are the first to be determined by micro-electron diffraction from a molecule of previously unknown structure,” noted Yifan Cheng at the University of California, San Francisco, in an accompanying Nature commentary. Others were similarly impressed. “To obtain a structure of this quality from a peptide material with such tiny crystals is a remarkable feat, and will probably serve as the model for many other studies,” Gregory Petsko at Weill Cornell Medical College in New York told Alzforum. Tim Bartels at Brigham and Women’s Hospital, Boston, agreed, “The resolution here is unprecedented.”
Meanwhile, in the September 8 Proceedings of the National Academy of Sciences, researchers led by Nikolaus Grigorieff at Janelia and Marcus Fändrich at Ulm University, Germany, describe a detailed structure for Aβ42 fibrils using electron cryomicroscopy. While cryoEM’s resolution is a tad lower than micro-electron diffraction's, this method allowed the researchers to examine the aggregated peptide in a more natural, hydrated state. Their model jibes with previous structural findings and explains some of the observed behavior of amyloid fibrils, commenters said.
It remains unknown whether the structures described in these papers occur in human disease. Researchers know that proteins aggregate into different strains, so the solved structures may represent but one possible configuration among many. Nonetheless, scientists expect these and future models to advance drug discovery. “The data will give medicinal chemists targets for designing therapeutic compounds,” noted David Teplow at UCLA. He was not involved in the work.
For many proteins, researchers obtain high-resolution structural data using nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. However, aggregated neurodegenerative proteins do not easily form the large crystals, or homogenous aggregates, these methods require. α-Synuclein, which accumulates in Parkinson’s disease, multiple-system atrophy (MSA), and other disorders, has been particularly recalcitrant. Previous spectroscopy studies hinted that the peptide contains β strands and that its fibril likely consists of β sheets, but the finer points were lacking (see Heise et al., 2005; Chen et al., 2007; Cho et al., 2011).
To get a closer look, Eisenberg and colleagues turned to micro-electron diffraction, a technique that combines features of X-ray crystallography and cryoEM. As in cryoEM, samples are frozen in a hydrated state, preserving their structure, then subjected to a beam of electrons in a transmission electron microscope. Unlike cyroEM, micro-electron diffraction does not directly produce an image of the sample, but instead analyzes a diffraction pattern, as does X-ray crystallography. This allows researchers to probe minute crystals, 10 billion times smaller than some of those used in X-ray crystallography, Eisenberg noted. By rotating these tiny crystals in the electron beam, researchers collect a series of diffraction patterns that reveal the three-dimensional structure of the protein. Previously, this method had been applied only to confirm known protein configurations.
Because full-length α-synuclein resists crystallization, joint first authors Jose Rodriguez, Magdalena Ivanova, Michael Sawaya, and Duilio Cascio at UCLA and Francis Reyes at Janelia crystallized a small segment comprising residues 68-78. This portion forms the core of a longer region known as NAC, or non-amyloid-β component. Other researchers have reported that this short “NACore,” as the authors call it, forms toxic amyloid fibrils on its own, while deleting the segment from α-synuclein prevents aggregation (see El-Agnaf et al., 1998; Periquet et al., 2007). The data imply that this NACore region controls aggregation of the full-length protein, the authors note. They aggregated a synthetic NACore fragment in vitro, obtaining crystals 50-300 nm across. Micro-electron diffraction then allowed them to visualize these crystals down to a resolution of 1.4 Å.
Diffraction data from the crystals supported a model in which two NACore strands dimerize tightly together, then stack up in layers to form two facing β sheets, similar to the configuration of other amyloid aggregates (see image above). Layers were separated by 4.8 Å and were staggered from each other, matching predictions from previous models. Surprisingly, however, the researchers found two water molecules nestled between the sheets in each layer. Most other β-sheet structures are dry. “This might imply that these α-synuclein fibers are not as ultra-stable as some other fibers, like Aβ,” Eisenberg suggested.
The researchers also crystallized and analyzed a separate segment of α-synuclein comprising residues 47-56 and containing the A53T mutation associated with early onset Parkinson’s disease. The mutated segment by itself formed stacked β sheets. Computer modeling predicted that in the full-length protein, the mutated 47-56 segment would fold over the NACore β sheets, making a thicker sandwich (see image at left). This additional binding interface might explain why α-synuclein containing the mutation aggregates more readily than wild-type versions, the authors suggested. Other familial Parkinson’s mutations, such as A30P and E46K, lie outside the studied regions. “[That] suggests that further structural surprises may be in store,” wrote Michel Goedert at the MRC Laboratory of Molecular Biology, Cambridge, U.K., in a Nature commentary.
How closely does the structure of the aggregated NACore mimic that of fibrils composed of full-length protein, as seen by others? Diffraction patterns from the two aggregates look roughly similar, hinting that the structures may be comparable. However, commenters noted that additional β strands present in the full-length protein might affect folding. In ongoing work, Eisenberg and colleagues are attempting to crystallize longer segments of α-synuclein to get a fuller picture of the fibrils.
How does this structure relate to reports of different structures or strains of α-synuclein (see Bousset et al., 2013; Peelaerts et al., 2015)? Ronald Melki at the French National Center for Scientific Research in the Paris suburb of Gif–sur–Yvette, noted that Eisenberg’s NACore structure matches the “ribbon” configuration of α-synuclein he recently described, which appears to associate more with MSA than PD (see Jun 2015 news; Sep 2015 news).
New Look at Aβ Fibrils
Grigorieff, Fändrich, and colleagues took a different tack to elucidate the structure of Aβ fibrils. They used standard cryoEM to bring fibrils into sharper focus than previous studies had allowed, achieving a resolution of about 7 Å. CryoEM does not require crystals; instead, researchers average images of several fibrils grown from the full-length peptide to build up a picture of the structure. Previously, the researchers used this technique to visualize Aβ40 fibrils. Those studies revealed structures composed of two protofilaments, each containing a paired β sheet at its core (see Sachse et al., 2008; Schmidt et al., 2009).
In the current study, joint first authors Matthias Schmidt at Ulm and Alexis Rohou at Janelia analyzed one type of synthetic Aβ42 aggregate made in vitro. A density map revealed two curved strands with their middle portions curling around each other (see image above). Computer modeling predicted that this structure was formed by the hydrophobic C-terminal domains of two Aβ42 peptides binding together in β strands, the side chains of their amino acids interdigitating in a “steric zipper.” Eisenberg had previously predicted this steric zipper based on X-ray crystallography of a small portion of Aβ, but not in the full fibril (see Sawaya et al., 2007).
Meanwhile, the charged N-terminal portions of each peptide splayed out to the sides. They were fuzzier, less resolved than the center, suggesting they move and assume multiple shapes. This jibes with previous data (see Dec 2010 conference news). The paired β strands of the peptides stack up to form sheets, composing the long axis of the fibril. The authors measured 10 Å between sheets, also in agreement with previous findings.
Other features conflicted with previous models. A recent NMR study, led by Yoshitaka Ishii at the University of Illinois in Chicago, reported that Aβ42 monomers fold up on themselves in an S-shape to form the core of amyloid fibrils (see May 2015 news). By contrast, Grigorieff and Fändrich’s model places an Aβ dimer at the heart of the fibril, with each peptide assuming a gently curving tilde structure in place of sharp S-bends. It is possible these models simply represent two different strains of fibril produced under distinct aggregation conditions, commenters noted. Likewise, a third model of the Aβ42 fibril, obtained by cryoEM, depicts two intertwined protofilaments around a hollow core (see Mar 2009 news). These latter fibrils were incubated at a highly acidic pH, unlike the physiological conditions used by Grigorieff and Fändrich.
Which, if any, of these structures predominate in Alzheimer’s brains remains to be determined. However, the new tilde-shaped model “accounts for a lot of prior observations [about Aβ], and follows basic principles of protein folding,” Teplow noted. For example, the greater propensity of Aβ42 than Aβ40 to aggregate could be attributed to the longer interface between β strands in the former (see image above). Also, the distinct shapes of the Aβ40 and Aβ42 fibrils could explain why the two peptides do not readily form mixed fibrils, the authors suggest.
The new model predicts that two charged residues at the edges of the dimer interface, Glu693 and Asp694, help keep the ends of the strands apart and prevent stronger binding. Mutations of these residues that remove the charge, such as the E693G Arctic or the D694N Iowa mutation, lead to early onset AD, perhaps due to increased aggregation. This suggests that the fibril shares some structural features with toxic peptide aggregates, Grigorieff noted. “The link between fibril structure and familial Alzheimer’s mutations hints that fibrils may play an important role in the disease,” he said.—Madolyn Bowman Rogers
- Shape of α-Synuclein Aggregates Influences Pathology
- α-Synuclein from Multiple System Atrophy Acts Like Prion in Mice
- San Diego: Flexible N-Termini Key to Aβ42 Oligomer Toxicity?
- Danger, S-Bends! New Structure for Aβ42 Fibrils Comes into View
- CryoEM Exposes Possible Achilles’ Heel in Aβ1-42 Fibrils
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- Watch Aβ Aggregate in Real Time Inside Cell’s Acid Vesicles
- Amyloid Fibrils Share a ‘Light’ Load
- Does Aβ Come In Strains? Glimpse Into Human Brain Suggests Yes
- Aβ Fibrils Drive Oligomer Formation, New Model Suggests
- Anti-parallel Universe—Rare Amyloid Peptides in Cylinders, Sheets
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