General structural motifs of amyloid protofilaments (original) (raw)
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Biochemistry, 2014
In Huntington's disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A "protective" C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (htt NT) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the βstructured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt βsheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the htt NT α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.
Biochemistry, 2010
The SH3 domain of the PI3 kinase (PI3-SH3 or PI3K-SH3) readily aggregates into fibrils in vitro and has served as an important model system to investigate the molecular properties and mechanism of formation of amyloid fibrils. We describe the molecular conformation of PI3-SH3 in amyloid fibril form as revealed by magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy. The MAS NMR spectra of these fibrils display excellent resolution, with narrow 13 C and 15 N line widths, representing a high degree of structural order and the absence of extensive molecular motion for the majority of the polypeptide chain. We have identified the spin-systems of 82 of the 86 residues in the protein, and obtained sequential resonance assignments for 75 of them. Chemical shift analysis indicates that the protein subunits making up the fibril adopt a compact conformation consisting of four well-defined β-sheet regions and four random-coil elements with varying degrees of local dynamics or disorder. The backbone conformation of PI3-SH3 in fibril form differs significantly from that of the native state of the protein, both in secondary structure and in the location of dynamic or disordered segments. The site-specific MAS NMR analysis of PI3-SH3 fibrils we report here is compared with previously published mechanistic and structural data, resulting in a detailed interpretation of the factors that mediate fibril formation by PI3-SH3 and allowing us to propose a possible model of the core structure of the fibrils. Our results confirm the structural similarities between PI3-SH3 fibrils and amyloids directly related to degenerative or infectious diseases. Amyloid fibrils are filamentous structures resulting from the spontaneous self-assembly of otherwise soluble peptides and proteins (1-4). A large number of human disorders, including Alzheimer's and Parkinson's diseases, type 2 diabetes and a variety of systemic amyloidoses, are associated with the formation of such macromolecular assemblies (1,5,6). In each of these pathological conditions, a specific peptide or protein, or protein fragment,
Unraveling the secrets of Alzheimer's beta-amyloid fibrils
Proceedings of the National Academy of Sciences of the United States of America, 2003
P roteins adopt an amazing array of sequence-dependent structures that enable them to perform the many chemical functions critical to life. Over the past decade, however, it has become clear that many different protein sequences can also form misfolded, insoluble aggregates known as amyloid fibrils, with common structural elements. Amyloid fibrils appear to be involved in a number of diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, prion diseases, and type II diabetes (1). Even ordinary globular proteins (e.g., myoglobin) can form amyloid fibrils under certain conditions (2), suggesting that fibril formation is a previously unappreciated general property of many proteins. Thus, it is of fundamental interest to understand how so many different protein sequences can adopt this alternative structure and it is of medical interest to understand and control disease-related amyloid formation. Knowledge of the three-dimensional structure of amyloid fibrils is critical for understanding the mechanism of fibrillogenesis and for design of possible inhibitors. Unfortunately, amyloid fibrils are noncrystalline and insoluble, and therefore are not amenable to x-ray crystallography and solution NMR, the classic tools of structural biology. In a recent issue of PNAS, Petkova et al. (3) report a structural model for Alzheimer's -amyloid fibrils deduced primarily from solid-state NMR experiments. This work provides a significant step forward in understanding -amyloid formation and showcases the power of solid-state NMR for obtaining structural information on important but challenging biomolecules. Amyloid fibrils share a number of characteristics, including a cross- structural motif (1). X-ray fiber diffraction data indicate that the fibrils contain -strands that are perpendicular to the fiber axis, with interstrand hydrogen bonding parallel to the fiber axis. However, fiber diffraction cannot determine the chemical details that are needed to understand fibrillogenesis, which parts of the sequence form the -strands and which specific amino acid residues are interacting, and cannot even determine whether the proteins in the fibril adopt a unique, ordered structure. This information has now been deduced by Petkova et al. for Alzheimer's -amy-See companion article on page 16742 in issue 26 of volume 99.
Rapid amyloid fiber formation from the fast-folding WW domain FBP28
Proceedings of the National Academy of Sciences, 2003
The WW domains are small proteins that contain a three-stranded, antiparallel -sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electrondiffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross- diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.
Structural Analysis of Alzheimer's β(1–40) Amyloid: Protofilament Assembly of Tubular Fibrils
Biophysical Journal, 1998
Detailed structural studies of amyloid fibrils can elucidate the way in which their constituent polypeptides are folded and self-assemble, and exert their neurotoxic effects in Alzheimer's disease (AD). We have previously reported that when aqueous solutions of the N-terminal hydrophilic peptides of AD -amyloid (A) are gradually dried in a 2-Tesla magnetic field, they form highly oriented fibrils that are well suited to x-ray fiber diffraction. The longer, more physiologically relevant sequences such as A(1-40) have not been amenable to such analysis, owing to their strong propensity to polymerize and aggregate before orientation is achieved. In seeking an efficient and inexpensive method for rapid screening of conditions that could lead to improved orientation of fibrils assembled from the longer peptides, we report here that the birefringence of a small drop of peptide solution can supply information related to the cooperative packing of amyloid fibers and their capacity for magnetic orientation. The samples were examined by electron microscopy (negative and positive staining) and x-ray diffraction. Negative staining showed a mixture of straight and twisted fibers. The average width of both types was ϳ70 Å, and the helical pitch of the latter was ϳ460 Å. Cross sections of plastic-embedded samples showed a ϳ60-Å-wide tubular structure. X-ray diffraction from these samples indicated a cross- fiber pattern, characterized by a strong meridional reflection at 4.74 Å and a broad equatorial reflection at 8.9 Å. Modeling studies suggested that tilted arrays of -strands constitute tubular, 30-Å-diameter protofilaments, and that three to five of these protofilaments constitute the A fiber. This type of structure-a multimeric array of protofilaments organized as a tubular fibril-resembles that formed by the shorter A fragments (e.g., A(6 -25), A(11-25), A(1-28)), suggesting a common structural motif in AD amyloid fibril organization.
Structural Characterization of GNNQQNY Amyloid Fibrils by Magic Angle Spinning NMR
Biochemistry, 2010
Several human diseases are associated with the formation of amyloid aggregates, but experimental characterization of these amyloid fibrils and their oligomeric precursors has remained challenging. Experimental and computational analysis of simpler model systems has therefore been necessary, for instance, on the peptide fragment GNNQQNY 7-13 of yeast prion protein Sup35p. Expanding on a previous publication, we report here a detailed structural characterization of GNNQQNY fibrils using magic angle spinning (MAS) NMR. On the basis of additional chemical shift assignments we confirm the coexistence of three distinct peptide conformations within the fibrillar samples, as reflected in substantial chemical shift differences. Backbone torsion angle measurements indicate that the basic structure of these coexisting conformers is an extended β-sheet. We structurally characterize a previously identified localized distortion of the β-strand backbone specific to one of the conformers. Intermolecular contacts are consistent with each of the conformers being present in its own parallel and in-register sheet. Overall the MAS NMR data indicate a substantial difference between the structure of the fibrillar and crystalline forms of these peptides, with a clearly increased complexity in the GNNQQNY fibril structure. These experimental data can provide guidance for future work, both experimental and theoretical, and provide insights into the distinction between fibril growth and crystal formation.
Journal of the …, 2011
Amyloid fibrils are high molecular weight aggregates formed by peptides and proteins with a characteristic cross-β structure in which β-sheets run parallel to the fibril axis. 1À3 A wide range of debilitating pathologies, including neurodegenerative disorders such as Alzheimer's disease and other conditions such as type 2 diabetes, involve amyloid fibrils and/or their precursor aggregates. 4 In addition, nonpathological and functional amyloid assemblies have been recognized, 5 and the observation of fibril formation by peptides and proteins unrelated to disease indicates that the amyloid fold is a generally accessible state of polypeptide chains. 3,4,6 There is therefore a very significant interest in deciphering the molecular architecture of amyloid fibrils and their precursors, from both the biomedical and the fundamental biophysical perspectives. The structures of proteins in amyloid fibrils differ conceptually from those of natively folded monomers. While the tertiary structure of monomers is the result of intramolecular forces, the structure in fibrils is typically determined by intermolecular interactions that give rise to the core β-sheet assembly. 7 In principle, the β-sheets in amyloid fibrils can be formed by parallel or antiparallel β-strands, or a combination of both, and with residues in or out of register between neighboring molecules. 8,9 The overall topology of amyloid fibrils is then defined by the 52 relative positions and orientations of the β-sheets that compose 53 the core of the fibril. 54 Despite the complexity of the molecular design of these 55 structures, magic-angle spinning nuclear magnetic resonance 56 (MAS NMR) studies have resulted in the elucidation of structur-57 al information relating to amyloid fibrils at the secondary structure 58 level via resonance assignment and chemical shift analysis 10À16 and 59 precise distance and torsion angle measurements. 17 In addition, 60 approximate distance constraints have been used to propose 61 models for various systems. 18À22 In the case of amyloid fibrils 62 formed by peptides amenable to solid-phase synthesis, the 63 tertiary structure can be probed by the incorporation of 13 C or 64 15 N labels at specific residues. A possible motif is a parallel, in-65 register arrangement of the β-sheets, which can be tested by 66 incorporation of a single 13 C label in all the molecules and the 67 measurement of 13 CÀ 13 C dipolar couplings. 8 These measure-68 ments are typically performed for various residues along the 69 sequence using separate samples and in one-dimensional (1D) 70 fashion. Several studies have utilized this and similar approaches, 71 such as inserting pairs of 13 C/ 13 C or 13 C/ 15 N nuclei, to derive
Dichotomous versus palm-type mechanisms of lateral assembly of amyloid fibrils
Protein Science, 2006
Despite possessing a common cross-β core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90–230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.
Structural complexity of a composite amyloid fibril
Journal of the American Chemical Society, 2011
The molecular structure of amyloid fibrils and the mechanism of their formation are of substantial medical and 9 biological importance, but present an ongoing experimental and computational challenge. An early high-resolution view of amyloidlike structure was obtained on amyloid-like crystals of a small fragment of the yeast prion protein Sup35p: the peptide GNNQQNY. As GNNQQNY also forms amyloid-like fibrils under similar conditions, it has been theorized that the crystal's structural features are shared by the fibrils. Here we apply magic-angle-spinning (MAS) NMR to examine the structure and dynamics of these fibrils. Previously multiple NMR signals were observed for such samples, seemingly consistent with the presence of polymorphic fibrils. Here we demonstrate that peptides with these three distinct conformations instead assemble together into composite protofilaments. Electron microscopy (EM) of the ribbon-like fibrils indicates that these protofilaments combine in differing ways to form striations of variable widths, presenting another level of structural complexity. Structural and dynamic NMR data reveal the presence of highly restricted side-chain conformations involved in interfaces between differently structured peptides, likely comprising interdigitated steric zippers. We outline molecular interfaces that are consistent with the observed EM and NMR data. The rigid and uniform structure of the GNNQQNY crystals is found to contrast distinctly with the more complex structural and dynamic nature of these "composite" amyloid fibrils. These results provide insight into the fibrilÀcrystal distinction and also indicate a necessary caution with respect to the extrapolation of crystal structures to the study of fibril structure and formation. 51 kinetics and thermodynamics of amyloid fibril formation, as well 52 as its stability and dye binding mechanisms. 3À21 Many of these 53 simulations employ the structure of the crystalline form of 54 the peptide as a reference point for the amyloid fibril structure, 55 despite recent experimental uncertainty about structural differ-56 ences or similarities between the crystals and fibrils. 22,23 57 Magic angle spinning (MAS) NMR is one of the few structural 58 methods that allows us to directly address this uncertainty as it 59 permits structural studies of fibrillar 24,25 as well as crystalline 60 peptides and proteins. 26À28 We had therefore previously initiated 61 MAS NMR-based structural studies of GNNQQNY as both 62 nanocrystals and amyloid-like fibrils. 22,29,30 63 One central observation made possible by the use of MAS was 64 that the fibrillar samples contained coexisting peptides in three 65 distinct molecular conformations. Such observations could be 66 explained by the presence of multiple fibril polymorphs, where 67 each conformation (as detected by NMR) would correspond to a 68 different macroscopic and structurally distinct fibril. 31,32 This type 69 of polymorphism, where GNNQQNY would seem able to adopt 70 two distinct crystalline forms and as many as three fibrillar forms, is 71 of significant interest. Polymorphic fibril formation has been 72 reported in vitro and in vivo and is thought to correlate for 73 instance to the strain phenomenon in prion diseases and to vari-74 able toxicities for different types of amyloid fibrils. 33 However, our 75 observations could also be accounted for by a different molecular 76 explanation: a single GNNQQNY fibril that contained multiple
Elongated Oligomers Assemble into Mammalian PrP Amyloid Fibrils
Journal of Molecular Biology, 2006
In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly a-helical state into b-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a b form of recombinant mouse PrP (residues 91-231). The b-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained threedimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of w60 Å. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-b amyloid fold.