13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

Siemer, Ansgar B.; Ritter, Christiane; Steinmetz, Michel O.; Ernst, Matthias; Riek, Roland; Meier, Beat H.

doi:10.1007/s10858-005-5582-7

Cited by 90 publications

(143 citation statements)

References 47 publications

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“…Nonetheless, our data for the Leu-labeled sample support the possibility that the M domain adopts a structure comprised of alternating ␤-strands (forming in-register parallel ␤-sheets) and loops, with the highly charged and proline-containing segments of the M domain being located in the loops. Similar alternation of ␤-strand segments and loop (or bend) segments in amyloid fibrils has been established for ␤-amyloid fibrils (23,32,44) and HET-s fibrils (33,45) and has been suggested for amylin fibrils (46), ␣-synuclein fibrils (36, 47), and Ure2p prion fibrils (20,48). The ␤-strand segments in these fibrils are typically 6-10 residues in length, whereas the loop segments may be only three to four residues in length and can in principle be much longer.…”

Section: Discussionsupporting

confidence: 65%

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Shewmaker¹,

Wickner²,

Tycko

2006

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

272

302

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The [PSI ؉ ] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup35 1-253 , consisting of the glutamine-and asparagine-rich Nterminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole-dipole couplings among 13 C nuclei in a series of Sup35NM fibril samples, 13 C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular 13 C-13 C distances of Ϸ0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of Ϸ0.5 nm. These results indicate that an in-register parallel ␤-sheet structure underlies the [PSI ؉ ] prion phenomenon. The Sup35p prion domain (Sup35N, residues 1-123) is asparagine-and glutamine-rich, is poor in charged residues, and has five imperfect nine-residue repeats with consensus YQQYN-PQGG. Sequence shuffling shows that the repeats are not necessary for prion generation or propagation and that amino acid content of the prion domain (not the sequence) determines whether a protein can form a prion (13). Certain point mutations in the prion domain can block propagation of [PSI ϩ ] introduced with the wild sequence (14, 15), although the mutant sequence may itself form a prion (16). Thus, propagation of an existing prion is very sequence-specific, as in the species barriers of mammalian prion diseases (reviewed in ref. 17).Amyloid fibrils are filamentous protein aggregates exhibiting ''cross-␤'' x-ray fiber diffraction patterns, indicating the presence of ␤-sheets formed by ␤-strands that are oriented approximately perpendicular to the fiber axis, with interstrand hydrogen bonds approximately parallel to the fiber axis (reviewed in ref. 18). The fact that the prion domains of Ure2p (another yeast prion protein with an N-terminal prion domain rich in asparagine and glutamine) and Sup35p can be shuffled and yet still form prions and amyloid (13,19) suggests that the amyloid on which these prions are based has an in-register parallel ␤-sheet structure (20, 21). A prion amyloid structure based on antiparallel ␤-sheets or ␤-helices would necessarily be stabilized by interactions among specific sets of unlike residues. These interactions would likely be destroyed by shuffling the sequence. In contrast, an in-register parallel ␤-sheet structure can be stabilized by intermolecular hydrophobic interactions (22,23) or polar side chain interactions [e.g., the ''polar zipper'' interactions suggested by Perutz et al. (24)] among like residues. Shuffling the sequence would still allow like residues to align and interact in such a structure. Thus, shuffleability of a prion domain suggests an in-register parallel ␤-sheet structure.The molecular structures of amyloid fibrils, particularly those formed by bona fide proteins, are difficult t...

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Section: Discussionsupporting

confidence: 65%

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Shewmaker¹,

Wickner²,

Tycko

2006

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

272

302

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“…The (so far exceptional) HETs prion domain amyloid is proposed to be an intramolecular ''pseudo'' parallel in-register ␤-sheet, but in some ways also resembles a ␤-helix (29,30).…”

Section: Discussionmentioning

confidence: 99%

“…The full-length A␤ 1-40 has a parallel in-register structure (25-27), as does amylin (17, 28), ␣-synuclein (18), and an amyloid of PrP (19). Solid-state NMR has also been used to obtain detailed information on the HETs protein prion domain (residues 218-289) (29,30), and residues 105-115 of transthyretin (31).The Ure2 and Sup35 prion proteins of yeast each consist of an N-terminal Q/N-rich amyloid-forming prion domain, a functional C-terminal domain and a connecting domain (refs. 32 and 33, reviewed in ref.…”

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confidence: 99%

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Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure

Wickner

Dyda²,

Tycko³

2008

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

139

130

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The [PIN ؉ ] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI ؉ ] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1⌬ strains have no known phenotype except for inability to propagate the [PIN ؉ ] prion. We used solidstate NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153-405) labeled with Tyr-1-13 C (14 residues), Leu-1-13 C (7 residues), or Ala-3-13 C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in ␤-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were Ϸ0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel ␤-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3-13 C indicate that the ␤-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN ؉ ]'s defining characteristic.T hree genetic criteria designed to identify infectious proteins [PIN ϩ ] has all of the genetic properties of a prion (4, 5), and was shown to be a self-propagating amyloid form of Rnq1p (4, 6), a protein rich in asparagine (N) and glutamine (Q), which had been shown to form self-propagating aggregates in vivo (7). Deletion of the RNQ1 gene produces no evident phenotype (7) (14), it is likely that variants of [PIN ϩ ] are different amyloid structures, although the precise nature of such differences remains unknown for any system at present.Aggregates formed in vitro from Rnq1p are filamentous and stain with thioflavin T (7). Circular dichroism indicates that these aggregates are high in ␤-sheet content and, on exposure to proteinase K, show partial resistance (6). These studies indicate that the Rnq1p aggregates are amyloid fibers. Moreover, these fibers formed in vitro from recombinant Rnq1p are infectious for yeast spheroplasts, efficiently transmitting the [PIN ϩ ] gene (6).Because amyloid is neither soluble nor crystalline, neither solution NMR nor x-ray crystallography is suitable for elucidating its detailed structure, although the study of amyloid-like microcrystals of short oligopeptides has revealed details of packing and sidechain interactions that are relevant to amyloid structures (15,16). Electron spin resonance studies have been used to study amylin (IAPP) and ␣-synuclein (17, 18) as well as amyloid of PrP (19). Solid-state NMR has been particularly useful for the elucidation of the structure of amyloids (reviewed in ref. 20). Selective labeling of particular amino acid residues with magnetic nuclear isotopes 13 C and 15 N, combined with pulse sequences designed...

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“…All assignments in Figure 5 can be considered tentative, in that they are based on comparisons with reported solution NMR chemical shifts. The high molecular weight of CA precludes direct sequential assignment by 2D and 3D solid state NMR techniques that have been applied to smaller proteins, 43,[59][60][61][62][63] for reasons of both resolution and sensitivity. Nonetheless, the good agreement between observed and expected crosspeak positions for a large number of crosspeaks indicates that the NTD and CTD structures studied by solution NMR are largely preserved in the CA assemblies.…”

Section: Spectral Simplification By Double-quantum Filteringmentioning

confidence: 99%

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

Chen

Tycko

2010

Protein Science

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The wild-type HIV-1 capsid protein (CA) self-assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV-1. Mass-per-length values of CA assemblies determined by dark-field transmission electron microscopy indicate a variety of structures, ranging from single-wall tubes to multiwall tubes that approximate solid rods. Two-dimensional (2D) solid state 13 CA 13 C and 15 NA 13 C NMR spectra of uniformly 15 N, 13 C-labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3-13 C 2 -glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N-terminal (NTD) and C-terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self-assembly of full-length CA into tubes. 2D 1 H-13 C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the a-helices of NTD and CTD and from the N-and C-terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.

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13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

Cited by 90 publications

References 47 publications

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

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13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

Cited by 90 publications

References 47 publications

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Amyloid of Rnq1p, the basis of the [ PIN + ] prion, has a parallel in-register β-sheet structure

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

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Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure