Witold K. Surewicz

Crystal structure of the human prion protein reveals a mechanism for oligomerization.

The pathogenesis of transmissible encephalopathies is associated with the conversion of the cellular prion protein, PrPC, into a conformationally altered oligomeric form, PrPSc. Here we report the crystal structure of the human prion protein in dimer form at 2 Å resolution. The dimer results from the three-dimensional swapping of the C-terminal helix 3 and rearrangement of the disulfide bond. An interchain two-stranded antiparallel β-sheet is formed at the dimer interface by residues that are located in helix 2 in the monomeric NMR structures. Familial prion disease mutations map to the regions directly involved in helix swapping. This crystal structure suggests that oligomerization through 3D domain-swapping may constitute an important step on the pathway of the PrPC → PrPSc conversion.

Acceleration of Amyloid Fibril Formation by Specific Binding of Aβ-(1–40) Peptide to Ganglioside-containing Membrane Vesicles

The interaction of Alzheimer’s Aβ peptide and its fluorescent analogue with membrane vesicles was studied by spectrofluorometry, Congo Red binding, and electron microscopy. The peptide binds selectively to the membranes containing gangliosides with a binding affinity ranging from 10−6 to 10−7 m depending on the type of ganglioside sugar moiety. This interaction appears to be ganglioside-specific as under our experimental conditions (neutral pH, physiologically relevant ionic strength), no Aβ binding was observed to ganglioside-free membranes containing zwitterionic or acidic phospholipids. Importantly, the addition of ganglioside-containing vesicles to the peptide solution dramatically accelerates the rate of fibril formation as compared with that of the peptide alone. The present results strongly suggest that the membrane-bound form of the peptide may act as a specific “template” (seed) that catalyzes the fibrillogenesis processin vivo.

Temperature-induced exposure of hydrophobic surfaces and its effect on the chaperone activity of α-crystallin

α‐Crystallin, the major protein of the ocular lens, is known to have extensive similarities to small heat shock proteins and to act as a molecular chaperone. The exposure of hydrophobic surfaces on α‐crystallin was studied by fluorescence spectroscopy using the hydrophobic probe bis‐ANS. Upon heating the protein undergoes a conformational transition which is associated with a marked increase in surface hydrophobicity. This transition, which occurs between approximately 38 and 5‐°C, lacks reversibility. The increase in surface hydrophobicity correlates with the increased chaperone activity of the protein. These results indicate that hydrophobic interactions play a major role in the chaperone action of α‐crystallin.

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and α-helical prion protein, PrPC, to the β-sheet-rich PrPSc. This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrPC monomer, structures of the pathogenic PrPSc or synthetic PrPSc-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrPC. Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrPC involves major refolding of the C-terminal α-helical region. The core of the amyloid maps to C-terminal residues from ≈160–220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of β-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to β-structure.

PH-DEPENDENT STABILITY AND CONFORMATION OF THE RECOMBINANT HUMAN PRION PROTEIN PRP(90-231)

A recombinant protein corresponding to the human prion protein domain encompassing residues 90–231 (huPrP(90–231)) was expressed in Escherichia coli in a soluble form and purified to homogeneity. Spectroscopic data indicate that the conformational properties and the folding pathway of huPrP(90–231) are strongly pH-dependent. Acidic pH induces a dramatic increase in the exposure of hydrophobic patches on the surface of the protein. At pH between 7 and 5, the unfolding of hPrP(90–231) in guanidine hydrochloride occurs as a two-state transition. This contrasts with the unfolding curves at lower pH values, which indicate a three-state transition, with the presence of a stable protein folding intermediate. While the secondary structure of the native huPrP(90–231) is largely α-helical, the stable intermediate is rich in β-sheet structure. These findings have important implications for understanding the initial events on the pathway toward the conversion of the normal into the pathological forms of prion protein.

Membrane environment alters the conformational structure of the recombinant human prion protein.

The prion protein (PrP) in a living cell is associated with cellular membranes. However, all previous biophysical studies with the recombinant prion protein have been performed in an aqueous solution. To determine the effect of a membrane environment on the conformational structure of PrP, we studied the interaction of the recombinant human prion protein with model lipid membranes. The protein was found to bind to acidic lipid-containing membrane vesicles. This interaction is pH-dependent and becomes particularly strong under acidic conditions. Spectroscopic data show that membrane binding of PrP results in a significant ordering of the N-terminal part of the molecule. The folded C-terminal domain, on the other hand, becomes destabilized upon binding to the membrane surface, especially at low pH. Overall, these results show that the conformational structure and stability of the recombinant human PrP in a membrane environment are substantially different from those of the free protein in solution. These observations have important implications for understanding the mechanism of the conversion between the normal (PrPC) and pathogenic (PrPSc) forms of prion protein.

Fibril Conformation as the Basis of Species- and Strain-Dependent Seeding Specificity of Mammalian Prion Amyloids

Spongiform encephalopathies are believed to be transmitted by self-perpetuating conformational conversion of the prion protein. It was shown recently that fundamental aspects of mammalian prion propagation can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop (PrP23-144). Here we demonstrate that PrP23-144 amyloids from different species adopt distinct secondary structures and morphologies, and that these structural differences are controlled by one or two residues in a critical region. These sequence-specific structural characteristics correlate strictly with the seeding specificity of amyloid fibrils. However, cross-seeding of PrP23-144 from one species with preformed fibrils from another species may overcome natural sequence-based structural preferences, resulting in a new amyloid strain that inherits the secondary structure and morphology of the template. These data provide direct biophysical evidence that protein conformations are transmitted in PrP amyloid strains, establishing a foundation for a structural basis of mammalian prion transmission barriers.

β-sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange

Propagation of transmissible spongiform encephalopathies is associated with the conversion of normal prion protein, PrPC, into a misfolded, oligomeric form, PrPSc. Although the high-resolution structure of the PrPC is well characterized, the structural properties of PrPSc remain elusive. Here we used MS analysis of H/D backbone amide exchange to examine the structure of amyloid fibrils formed by the recombinant human PrP corresponding to residues 90–231 (PrP90–231), a misfolded form recently reported to be infectious in transgenic mice overexpressing PrPC. Analysis of H/D exchange data allowed us to map the systematically H-bonded β-sheet core of PrP amyloid to the C-terminal region (staring at residue ≈169) that in the native structure of PrP monomer corresponds to α-helix 2, a major part of α-helix 3, and the loop between these two helices. No extensive hydrogen bonding (as indicated by the lack of significant protection of amide hydrogens) was detected in the N-terminal part of PrP90–231 fibrils, arguing against the involvement of residues within this region in stable β-structure. These data provide long-sought experimentally derived constraints for high-resolution structural models of PrP amyloid fibrils.

Familial Mutations and the Thermodynamic Stability of the Recombinant Human Prion Protein

Hereditary forms of human prion disease are linked to specific mutations in the PRNP gene. It has been postulated that these mutations may facilitate the pathogenic process by reducing the stability of the prion protein (PrP). To test this hypothesis, we characterized the recombinant variants of human PrP(90–231) containing point mutations corresponding to Gerstmann-Straussler-Scheinker disease (P102L), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (M129/D178N). The first two of these mutants could be recovered form from the periplasmic space of Escherichia coli in a soluble form, whereas the D178N variant aggregated into inclusion bodies. The secondary structure of the two soluble variants was essentially identical to that of the wild-type protein. The thermodynamic stability of these mutants was assessed by unfolding in guanidine hydrochloride and thermal denaturation. The stability properties of the P102L variant were indistinguishable from those of wild-type PrP, whereas the E200K mutation resulted in a very small destabilization of the protein. These data, together with the predictive analysis of other familial mutations, indicate that some hereditary forms of prion disease cannot be rationalized using the concept of mutation-induced thermodynamic destabilization of the cellular prion protein.

Mammalian Prions Generated from Bacterially Expressed Prion Protein in the Absence of Any Mammalian Cofactors

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrPC, to a misfolded aggregated form, PrPSc. The protein-only hypothesis asserts that PrPSc itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.