X-ray Fiber Diffraction and Computational Analyses of Stacked Hexads in Supramolecular Polymers: Insight into Self-Assembly in Water by Prospective Prebiotic Nucleobases.

Aqueous solutions of equimolar mixtures of 2,4,6-triaminopyrimidine (TAP) and carboxylic acid substituted cyanuric acid (CyCo6 or R-4MeCyCo6) monomers self-assemble into gel-forming supramolecular polymers. Macroscopic fibers drawn from these mixtures were analyzed by X-ray diffraction to determine their molecular structures. Computational methods were used to explore the intrinsic intermolecular interactions that contribute to the structure and stability of these assemblies. Both polymers are formed by the stacking of hexameric rosettes, (TAP/CyCo6)3 or (TAP/R-4MeCyCo6)3, respectively, into long, stiff, twisted stacks of essentially planar rosettes. Chiral, left-handed supramolecular polymers with a helical twist angle of -26.7° per hexad are formed when the pure enantiomer R-4MeCyCo6 is used. These hexad stacks pack into bundles with a hexagonal crystalline lattice organization perpendicular to the axis of the macroscopic fiber. Polymers formed from TAP and CyCo6, both of which are achiral, assemble into macroscopic domains that are packed as a centered rectangular lattice. Within these domains, the individual polymers exist as either right-handed or left-handed helical stacks, with twist angles of +15° or -15° per hexad, respectively. The remarkable ability of TAP and cyanuric acid derivatives to self-assemble in water, and the structural features of their supramolecular polymers reported here, provide additional support for the proposal that these heterocycles could have served as recognition units for an early form of nucleic acids, before the emergence of RNA.

Fungal prion HET-s as a model for structural complexity and self-propagation in prions.

The highly ordered and reproducible structure of the fungal prion HET-s makes it an excellent model system for studying the inherent properties of prions, self-propagating infectious proteins that have been implicated in a number of fatal diseases. In particular, the HET-s prion-forming domain readily folds into a relatively complex two-rung ß-solenoid amyloid. The faithful self-propagation of this fold involves a diverse array of inter- and intramolecular structural features. These features include a long flexible loop connecting the two rungs, buried polar residues, salt bridges, and asparagine ladders. We have used site-directed mutagenesis and X-ray fiber diffraction to probe the relative importance of these features for the formation of ß-solenoid structure, as well as the cumulative effects of multiple mutations. Using fibrillization kinetics and chemical stability assays, we have determined the biophysical effects of our mutations on the assembly and stability of the prion-forming domain. We have found that a diversity of structural features provides a level of redundancy that allows robust folding and stability even in the face of significant sequence alterations and suboptimal environmental conditions. Our findings provide fundamental insights into the structural interactions necessary for self-propagation. Propagation of prion structure seems to require an obligatory level of complexity that may not be reproducible in short peptide models.

Structural studies of truncated forms of the prion protein PrP.

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrP(Sc).

Fiber diffraction of the prion-forming domain HET-s(218-289) shows dehydration-induced deformation of a complex amyloid structure.

Amyloids are filamentous protein aggregates that can be formed by many different proteins and are associated with both disease and biological functions. The pathogenicities or biological functions of amyloids are determined by their particular molecular structures, making accurate structural models a requirement for understanding their biological effects. One potential factor that can affect amyloid structures is hydration. Previous studies of simple stacked ß-sheet amyloids have suggested that dehydration does not impact structure, but other studies indicated dehydration-related structural changes of a putative water-filled nanotube. Our results show that dehydration significantly affects the molecular structure of the fungal prion-forming domain HET-s(218-289), which forms a ß-solenoid with no internal solvent-accessible regions. The dehydration-related structural deformation of HET-s(218-289) indicates that water can play a significant role in complex amyloid structures, even when no obvious water-accessible cavities are present.

Heterogeneous seeding of a prion structure by a generic amyloid form of the fungal prion-forming domain HET-s(218-289).

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung ß-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked ß-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked ß-sheet fibrils nucleate the formation of the infectious ß-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious ß-solenoid fibrils) fibrillization. Several serial passages of stacked ß-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

Is supramolecular filament chirality the underlying cause of major morphology differences in amyloid fibrils?

The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for "normal" left-hand-helical filaments and below pH 2 for "reversed" right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-ß-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218-289) prion, and a short polypeptide fragment of transthyretin, TTR (105-115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases.

Degradation of fungal prion HET-s(218-289) induces formation of a generic amyloid fold.

The prion-forming domain of the fungal prion protein HET-s, HET-s(218-289), is known from solid-state NMR studies to have a ß-solenoidal structure; the ß-solenoid has the cross-ß structure characteristic of all amyloids, but is inherently more complex than the generic stacked ß-sheets found in studies of small synthetic peptides. At low pH HET-s(218-289) has also been reported to form an alternative structure, which has not been characterized. We have confirmed by x-ray fiber diffraction that HET-s(218-289) adopts a ß-solenoidal structure at neutral pH, and shown that at low pH, it forms either a ß-solenoid or a stacked ß-sheet structure, depending on the integrity of the protein and the conditions of fibrillization. The low pH stacked-sheet structure is usually formed only by proteolyzed HET-s(218-289), but intact HET-s(218-289) can form stacked sheets when seeded with proteolyzed stacked-sheet HET-s(218-289). The polymorphism of HET-s parallels the structural differences between the infectious brain-derived and the much less infectious recombinant mammalian prion protein PrP. Taken together, these observations suggest that the functional or pathological forms of amyloid proteins are more complex than the simple generic stacked-sheet amyloids commonly formed by short peptides.

Artificial forisomes are ideal models of forisome assembly and activity that allow the development of technical devices.

Forisomes are protein polymers found in leguminous plants that have the remarkable ability to undergo reversible "muscle-like" contractions in the presence of divalent cations and in extreme pH environments. To gain insight into the molecular basis of forisome structure and assembly, we used confocal laser scanning microscopy to monitor the assembly of fluorescence-labeled artificial forisomes in real time, revealing two distinct assembly processes involving either fiber elongation or fiber alignment. We also used scanning and transmission electron microscopy and X-ray diffraction to investigate the ultrastructure of forisomes, finding that individual fibers are arranged into compact fibril bundles that disentangle with minimal residual order in the presence of calcium ions. To demonstrate the potential applications of artificial forisomes, we created hybrid protein bodies from forisome subunits fused to the B-domain of staphylococcal protein A. This allowed the functionalization of the artificial forisomes with antibodies that were then used to target forisomes to specific regions on a substrate, providing a straightforward approach to develop forisome-based technical devices with precise configurations. The functional contractile properties of forisomes are also better preserved when they are immobilized via affinity reagents rather than by direct contact to the substrate. Artificial forisomes produced in plants and yeast therefore provide an ideal model for the investigation of forisome structure and assembly and for the design and testing of tailored artificial forisomes for technical applications.

Helical viruses.

Virtually all studies of structure and assembly of viral filaments have been made on plant and bacterial viruses. Structures have been determined using fiber diffraction methods at high enough resolution to construct reliable molecular models or several of the rigid plant tobamoviruses (related to tobacco mosaic virus, TMV) and the filamentous bacteriophages including Pf1 and fd. Lower-resolution structures have been determined for a number of flexible filamentous plant viruses using fiber diffraction and cryo-electron microscopy. Virions of filamentous viruses have numerous mechanical functions, including cell entry, viral disassembly, viral assembly, and cell exit. The plant viruses, which infect multicellular organisms, also use virions or virion-like assemblies for transport within the host. Plant viruses are generally self-assembling; filamentous bacteriophage assembly is combined with secretion from the host cell, using a complex molecular machine. Tobamoviruses and other plant viruses disassemble concomitantly with translation, by various mechanisms and involving various viral and host assemblies. Plant virus movement within the host also makes use of a variety of viral proteins and modified host assemblies.

Surface charge of polyoxometalates modulates polymerization of the scrapie prion protein.

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). N-terminally truncated PrP(Sc), denoted PrP 27-30, retains infectivity and polymerizes into rods with the ultrastructural and tinctorial properties of amyloid. We report here that some polyoxometalates (POMs) favor polymerization of PrP 27-30 into prion rods, whereas other POMs promote assembly of the protein into 2D crystals. Antibodies reacting with epitopes in denatured PrP 27-30 also bound to 2D crystals treated with 3 M urea. These same antibodies did not bind to either native PrP(Sc) or untreated 2D crystals. By using small, spherical POMs with Keggin-type structures, the central heteroatom was found to determine whether prion rods or 2D crystals were preferentially formed. An example of a Keggin-type POM with a phosphorous heteroatom is the phosphotungstate anion (PTA). Both PTA and a Keggin-type POM with a silicon heteratom have low-charge densities and favor formation of prion rods. In contrast, POMs with boron or hydrogen heteroatoms exhibiting higher negative charges encouraged 2D crystal formation. The 2D crystals of PrP 27-30 produced by selective precipitation with POMs were larger and more well ordered than those obtained by sucrose gradient centrifugation. Our findings argue that the negative charge of Keggin-type POMs determines the quaternary structure adopted by PrP 27-30. The mechanism by which POMs function in competing prion polymerization pathways--one favoring 2D crystals and the other, amyloid fibrils--remains to be established.

Natural and synthetic prion structure from X-ray fiber diffraction.

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Enhanced purification coupled with biophysical analyses shows cross-ß structure as a core building block for Streptococcus mutans functional amyloids.

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-ß amyloid structure.

Structure of flexible filamentous plant viruses.

Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.

Structural Biology of PrP Prions.

Prion diseases are characterized by the deposition of amyloids, misfolded conformers of the prion protein. The misfolded conformation is self-replicating, by a mechanism solely enciphered in the conformation of the protein. Because of low solubility and heterogeneous aggregate sizes, the detailed atomic structure of the infectious isoform is still unknown. Progress has, however, been made, and has allowed insights into the structural and disease-related mechanisms of prions. Many structural models have been proposed, and a number of them support a consensus trimeric ß-helical model, significantly more complex than simple amyloid models. There is evidence that such complexity may be a necessary property of prion structure. Knowledge of the structure of prions will provide a greater understanding of the protein isoform conversion mechanism, and could eventually lead to rationally designed intervention strategies.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein.

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register ß-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

Rapid Filament Supramolecular Chirality Reversal of HET-s (218-289) Prion Fibrils Driven by pH Elevation.

Amyloid fibril polymorphism is not well understood despite its potential importance for biological activity and associated toxicity. Controlling the polymorphism of mature fibrils including their morphology and supramolecular chirality by postfibrillation changes in the local environment is the subject of this study. Specifically, the effect of pH on the stability and dynamics of HET-s (218-289) prion fibrils has been determined through the use of vibrational circular dichroism (VCD), deep UV resonance Raman, and fluorescence spectroscopies. It was found that a change in solution pH causes deprotonation of Asp and Glu amino acid residues on the surface of HET-s (218-289) prion fibrils and triggers rapid transformation of one supramolecular chiral polymorph into another. This process involves changes in higher order arrangements like lateral filament and fibril association and their supramolecular chirality, while the fibril cross-ß core remains intact. This work suggests a hypothetical mechanism for HET-s (218-289) prion fibril refolding and proposes that the interconversion between fibril polymorphs driven by the solution environment change is a general property of amyloid fibrils.

Heterogeneous seeding of HET-s(218-289) and the mutability of prion structures.

One fundamental property of prions is the formation of strains-prions that have distinct biological effects, despite a common amino acid sequence. The strain phenomenon is thought to be caused by the formation of different molecular structures, each encoding for a particular biological activity. While the precise mechanism of the formation of strains is unknown, they tend to arise following environmental changes, such as passage between different species. One possible mechanism discussed here is heterogeneous seeding; the formation of a prion nucleated by a different molecular structure. While heterogeneous seeding is not the only mechanism of prion mutation, it is consistent with some observations on species adaptation and drug resistance. Heterogeneous seeding provides a useful framework to understand how prions can adapt to new environmental conditions and change biological phenotypes.

Barley stripe mosaic virus: structure and relationship to the tobamoviruses.

Barley stripe mosaic virus (BSMV) is the type member of the genus Hordeivirus, rigid, rod-shaped viruses in the family Virgaviridae. We have used fiber diffraction and cryo-electron microscopy to determine the helical symmetry of BSMV to be 23.2 subunits per turn of the viral helix, and to obtain a low-resolution model of the virus by helical reconstruction methods. Features in the model support a structural relationship between the coat proteins of the hordeiviruses and the tobamoviruses.