Insight into the PrPC-->PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants.

Prion diseases are associated with the conversion of the alpha-helix rich prion protein (PrPC) into a beta-structure-rich insoluble conformer (PrPSc) that is thought to be infectious. The mechanism for the PrPC-->PrPSc conversion and its relationship with the pathological effects of prion diseases are poorly understood, partly because of our limited knowledge of the structure of PrPSc. In particular, the way in which mutations in the PRNP gene yield variants that confer different susceptibilities to disease needs to be clarified. We report here the 2.5-A-resolution crystal structures of three scrapie-susceptibility ovine PrP variants complexed with an antibody that binds to PrPC and to PrPSc; they identify two important features of the PrPC-->PrPSc conversion. First, the epitope of the antibody mainly consists of the last two turns of ovine PrP second alpha-helix. We show that this is a structural invariant in the PrPC-->PrPSc conversion; taken together with biochemical data, this leads to a model of the conformational change in which the two PrPC C-terminal alpha-helices are conserved in PrPSc, whereas secondary structure changes are located in the N-terminal alpha-helix. Second, comparison of the structures of scrapie-sensitivity variants defines local changes in distant parts of the protein that account for the observed differences of PrPC stability, resistant variants being destabilized compared with sensitive ones. Additive contributions of these sensitivity-modulating mutations to resistance suggest a possible causal relationship between scrapie resistance and lowered stability of the PrP protein.

[1]  M. Moudjou,et al.  Cellular prion protein status in sheep: tissue-specific biochemical signatures. , 2001, The Journal of general virology.

[2]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[3]  M. Beekes,et al.  Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method. , 1995, The Journal of general virology.

[4]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[5]  Richard C. Moore,et al.  Mice with gene targetted prion protein alterations show that Prnp, Sine and Prni are congruent , 1998, Nature Genetics.

[6]  A. Shrake,et al.  Environment and exposure to solvent of protein atoms. Lysozyme and insulin. , 1973, Journal of molecular biology.

[7]  Vincent C. Emery,et al.  J. Virol. Methods , 1996 .

[8]  D. Calavas,et al.  Sensitivity of the Western blot detection of prion protein PrPres in natural sheep scrapie. , 1998, Journal of virological methods.

[9]  R. Riek,et al.  NMR structure of the mouse prion protein domain PrP(121–231) , 1996, Nature.

[10]  H. Scheraga,et al.  Intramolecular Versus Intermolecular Disulfide Bonds in Prion Proteins* , 2002, The Journal of Biological Chemistry.

[11]  J. Grosclaude,et al.  High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility. , 2000, European journal of biochemistry.

[12]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[13]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[14]  P. Bayley,et al.  The crystal structure of the globular domain of sheep prion protein. , 2004, Journal of molecular biology.

[15]  K Wüthrich,et al.  NMR structure of the bovine prion protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  F. Cohen,et al.  Dominant-negative inhibition of prion replication in transgenic mice , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  F. Heppner,et al.  Prevention of Scrapie Pathogenesis by Transgenic Expression of Anti-Prion Protein Antibodies , 2001, Science.

[18]  J. Grosclaude,et al.  Amyloidogenic unfolding intermediates differentiate sheep prion protein variants. , 2002, Journal of molecular biology.

[19]  K. Beyreuther,et al.  Two alleles of a neural protein gene linked to scrapie in sheep. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  R. Glockshuber,et al.  Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. , 1999, Biochemistry.

[22]  A. Aguzzi,et al.  Mice devoid of PrP are resistant to scrapie , 1993, Cell.

[23]  A. Mazur,et al.  Sheep Prion Protein Synthetic Peptide Spanning Helix 1 and β-Strand 2 (Residues 142–166) Shows β-Hairpin Structure in Solution* , 2001, The Journal of Biological Chemistry.

[24]  K Wüthrich,et al.  NMR structures of three single-residue variants of the human prion protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  F. Cohen,et al.  Solution structure of Syrian hamster prion protein rPrP(90-231). , 1999, Biochemistry.

[26]  Witold K. Surewicz,et al.  Crystal structure of the human prion protein reveals a mechanism for oligomerization , 2002, Nature Structural Biology.

[27]  Stanley B. Prusiner,et al.  Nobel Lecture: Prions , 1998 .

[28]  V. Luzzati,et al.  Traitement statistique des erreurs dans la determination des structures cristallines , 1952 .

[29]  R. Zahn,et al.  Influence of pH on NMR Structure and Stability of the Human Prion Protein Globular Domain* , 2003, Journal of Biological Chemistry.

[30]  J. Collinge,et al.  Monoclonal antibodies inhibit prion replication and delay the development of prion disease , 2003, Nature.

[31]  J. Rossier,et al.  Transmission of the BSE Agent to Mice in the Absence of Detectable Abnormal Prion Protein , 1997, Science.

[32]  S. Laboissiere,et al.  A prion protein epitope selective for the pathologically misfolded conformation , 2003, Nature Medicine.

[33]  J. Kelly,et al.  The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. , 1998, Current opinion in structural biology.

[34]  R J Fletterick,et al.  Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Z. Eshhar,et al.  X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[36]  P E Wright,et al.  Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  H. Laude,et al.  PrP Polymorphisms Tightly Control Sheep Prion Replication in Cultured Cells , 2003, Journal of Virology.

[38]  R. Glockshuber,et al.  Folding and intrinsic stability of deletion variants of PrP(121-231), the folded C-terminal domain of the prion protein. , 2002, Biophysical chemistry.

[39]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[40]  N. Hunter,et al.  Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. , 1996, The Journal of general virology.

[41]  F E Cohen,et al.  A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform. , 1997, Journal of molecular biology.

[42]  David A Agard,et al.  Structural studies of the scrapie prion protein by electron crystallography , 2002, Proceedings of the National Academy of Sciences of the United States of America.