Wen Bian

Natural and synthetic prion structure from X-ray fiber diffraction

A conformational isoform of the mammalian prion protein (PrPSc) 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-β diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of β 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 β-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 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked β-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.

Structure of Flexible Filamentous Plant Viruses

ABSTRACT 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.

WCEN : a computer program for initial processing of fiber diffraction patterns

Processing of fiber diffraction patterns is generally more difficult than for single-crystal patterns, and requires different algorithms and software. The program WCEN has been developed to determine experimental and specimen parameters and to convert diffraction data from detector to reciprocal space, and offers a variety of input and output formats, running under Mac OS X and Linux. The program is described and examples from oriented sols of filamentous plant viruses, illustrating different strategies for parameter determination and refinement, are given.

Fiber diffraction data indicate a hollow core for the Alzheimer's aβ 3-fold symmetric fibril.

Amyloid β protein (Aβ), the principal component of the extracellular plaques found in the brains of patients with Alzheimers disease, forms fibrils well suited to structural study by X-ray fiber diffraction. Fiber diffraction patterns from the 40-residue form Aβ(1-40) confirm a number of features of a 3-fold symmetric Aβ model from solid-state NMR (ssNMR) but suggest that the fibrils have a hollow core not present in the original ssNMR models. Diffraction patterns calculated from a revised 3-fold hollow model with a more regular β-sheet structure are in much better agreement with the observed diffraction data than patterns calculated from the original ssNMR model. Refinement of a hollow-core model against ssNMR data led to a revised ssNMR model, similar to the fiber diffraction model.

A Common Structure for the Potexviruses

We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to confirm the symmetry of three potexviruses, potato virus X, papaya mosaic virus, and narcissus mosaic virus, and to determine their low-resolution structures. All three viruses have slightly less than nine subunits per turn of the viral helix. Our data strongly support the view that all potexviruses have approximately the same symmetry. The structures are dominated by a large domain at high radius in the virion, with a smaller domain, which includes the putative RNA-binding site, extending to low radius.

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).

Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289)

Background: Prions are aberrantly folded infectious proteins whose biological activity is determined by their distinct folds. Results: The infectious fold of the fungal prion-forming domain HET-s(218–289) can be nucleated by a noninfectious polymorph. Conclusion: Generic amyloid architectures can seed the formation of infectious prions. Significance: Heterogeneous seeding may be a mechanism of prion strain adaptation and interspecies transmission. 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 β-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 β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-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.

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.

Architecture of the potyviruses

X-ray fiber diffraction data were obtained and helical pitch and symmetry were determined for seven members of the family Potyviridae, including representatives from the genera Potyvirus, Rymovirus, and Tritimovirus. The diffraction patterns are similar, as expected. There are, however, significant variations in the symmetries, as previously found among the flexible potexviruses, but not among the rigid tobamoviruses. Wheat streak mosaic virus, the only member of the genus Tritimovirus examined, displayed the largest deviations in diffraction data and helical parameters from the other viruses in the group.

Flexible filamentous virus structures from fiber diffraction

Fiber diffraction data have been obtained from Narcissus mosaic virus, a potexvirus from the family Flexiviridae, and soybean mosaic virus (SMV), a potyvirus from the family Potyviridae. Analysis of the data in conjunction with cryo-electron microscopy data allowed us to determine the symmetry of the viruses and to make reconstructions of SMV at 19 {angstrom} resolution and of another potexvirus, papaya mosaic virus, at 18 {angstrom} resolution. These data include the first well-ordered data ever obtained for the potyviruses and the best-ordered data from the potexviruses, and offer the promise of eventual high resolution structure determinations.