Atomic‐Resolution Three‐Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation

Schütz, Anne K.; Vagt, Toni; Huber, Matthias; Ovchinnikova, Oxana; Cadalbert, Riccardo; Wall, Joseph S.; Güntert, Peter; Böckmann, Anja; Glockshuber, Rudi; Meier, Beat H.

doi:10.1002/anie.201408598

Cited by 258 publications

(369 citation statements)

References 36 publications

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“…1). To do so, we prepared chimeric His 6 -NM[ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) ]-Npu[ (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) ]-His 6 , expressed in natural-abundance media (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

“…The final product was greater than 90% pure by SDS/PAGE and contained a single cysteine mutation (or "scar") at the ligation site. We prepared more than 8 mg of segmentally labeled NM molecules per liter of isotopically labeled growth for a practical yield of about 50% of the His 6 -NM[ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) ]-DnaE[ ] precursor. To avoid sample heterogeneity derived from multiple fiber forms, the segmentally labeled NM was templated into amyloid fibers using amyloid seeds derived from lysates of yeast carrying the strong [PSI + ] phenotype (11).…”

Section: Resultsmentioning

confidence: 99%

“…These differences are smaller than the experimental error in our assignments. (10). The approach is simple.…”

Section: Resultsmentioning

confidence: 99%

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Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C-and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. (2), is an amyloid form of the translation termination factor Sup35 (3). The amyloid fold is polymeric fiber of protein monomers that is rich in β-sheets that run perpendicular to the fiber axis. Amyloid formation sequesters Sup35 from the ribosome, changing the rate of stop-codon read-through and creating a variety of new yeast phenotypes (4,5). Different regions of the Sup35 protein are responsible for prion templating, prion inheritance, and translation termination. The N-terminal domain "N" is Q/N-rich and required for the formation and templating of amyloid. The highly charged K/E-rich middle domain "M" promotes solubility in the nonprion form and is required for chaperone-mediated prion inheritance (6, 7). The C-terminal domain is necessary and sufficient for translation termination. The N and M domains together, NM, contain all of the necessary features to act as an amyloid-based prion.A wide variety of approaches have been used to investigate Sup35 prion structures, but a consensus structural picture has yet to emerge. Amyloids are notoriously difficult to study, mainly because they are insoluble yet noncrystalline and are therefore not easily amenable to traditional structural biology methods. In all models, at least some part of the N domain is involved in the amyloid fold (8-11), and most of the M domain is thought to be solvent-accessible and intrinsically disordered (11-13). However, there are two conflicting models of how monomers of NM are arranged in amyloid fibers. In one model, called the β-helical model, monomers make intermolecular contacts in discrete regions, with the region in-between making intramolecular contacts. This model is supported by the patterns of interaction and cross-linking determined from a series of site-directed mutants (8). The work has the caveat that mutations may perturb the fibril structure. However, fibers made from the mutated versions of the protein ca...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…This motif was previously seen by X-ray crystallography in microcrystals encompassing steric zipper structures, and by cryo-EM in the fibrils formed from AL1, Aβ , and Aβ (1-42) peptides (7,9). Furthermore, there is solid-state NMR evidence for peptide dimers in fibrils formed from a transthyretin-derived peptide fragment (11), an N-terminally extended variant of Aβ (1-40) (28), and from an Aβ peptide variant carrying the Osaka mutation (29). The peptide dimer motif was also found in several structural models of longer polypeptide chains, such as ribonuclease A (16,17,30).…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril

Schmidt

Annamalai

Schmidt

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles.

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“…Notably, in this context, the functional nontoxic HET-s prion amyloid has a hydrophilic surface typically observed for soluble proteins (HET-s(218-289) overexpression, both in its normal host Podospora anserina and in rat, is not toxic) (Fig. 2) (Winner et al 2011), whereas the disease-associated Ab amyloids appear to have significant hydrophobic patches on their surfaces with the potential for nonspecific hydrophobic interactions with other proteins or membranes, which may cause toxicity (Lu et al 2013;Schütz et al 2015).…”

Section: D Structures Of Amyloidsmentioning

confidence: 99%

The Three-Dimensional Structures of Amyloids

Riek

2016

Cold Spring Harb Perspect Biol

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Amyloids are highly ordered protein aggregates that are associated with both disease (including PrP prion, Alzheimer's, and Parkinson's) and biological function. The amyloid structure is composed of the cross-b-sheet entity, which is an almost indefinitely repeating twolayered intermolecular b-sheet motif. The three-dimensional (3D) structure is unique among protein folds because it folds only upon intermolecular contacts (for a folding to occur, only short sequences of amino acid residues are required), and the structure repeats itself at the atomic level (i.e., every 4.7 Å ). As a consequence of this structure, among others, it can grow by recruiting corresponding amyloid peptide/protein and thus has the capacity to be an infectious protein (i.e., a prion). Furthermore, its repetitiveness can translate what would be a nonspecific activity as monomer into a potent one through cooperativity. Because of these and other properties, the activities of amyloids are manifold and include peptide storage, template assistance, loss of function, gain of function, generation of toxicity, membrane binding, infectivity, and more. This review summarizes the structural nature of the cross-bsheet motif on the basis of a few high-resolution structural studies of amyloids in the context of potential biological activities.

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Atomic‐Resolution Three‐Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation

Cited by 258 publications

References 36 publications

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril

The Three-Dimensional Structures of Amyloids

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