Simone Hornemann and Rudi Glockshuber J. Mol. Biol. (1996) 262, 614‚619 (Received 8 May 1996; received in revised form 31 May 1996; accepted 26 June 1996)Prion diseases are assumed to be caused by the infectious isoform, PrP Sc , of a single cellular surface protein, PrP C . PrP Sc is an insoluble form of PrP C and is believed to possess a different three-dimensional fold. It may propagate by causing PrP C to adopt its own infectious conformation by an unknown mechanism. Studies on folding and thermodynamic stability of prion proteins are essential for understanding the processes underlying the conversion from PrP C to PrP Sc , but have so far been hampered by the low solubility of prion proteins in the absence of detergents. Here, we show that the amino-terminally truncated segment of mouse PrP comprising residues 121 to 231 is an autonomous folding unit. It consists predominantly of a-helical secondary structure and is soluble at high concentrations up to 1 mM in distilled water. PrP(121-231) undergoes a cooperative and completely reversible unfolding/refolding transition in the presence of guanidinium chloride with a free energy of folding of -22 kJ/mol at pH 7. The intrinsic stability of segment 121-231 is not in accordance with present models of the structure of PrP C and PrP Sc. PrP(121-231) may represent the only part of PrP C with defined three-dimensional structure.
The ååprotein only¼¼ hypothesis (Griffith, 1967) has turned out to be the most suitable working hypothesis for investigating the processes involved in the development and transmission of prion diseases (transmissible spongiform encephalopathies). It proposes that the infectious agent, the prion, is a single protein, termed PrP Sc . PrP Sc appears to represent a modified form of a natural cellular surface protein of the host, PrP C (Prusiner, 1982). PrP Sc is likely to be chemically identical with PrP C (Stahl & Prusiner, 1991), but may possess a different three-dimensional structure (Pan et al., 1993). It may multiply by a PrP Sc -catalyzed conformational transition from PrP C to PrP Sc (Prusiner, 1991). A large body of evidence including in vivo as well as in vitro experiments has been accumulated in the last few years supporting the protein only hypothesis (Prusiner, 1991; Weissmann, 1994, 1995). The known sequences of mammalian prion proteins are strikingly similar and pairs of sequences are generally more than 90% identical (Scha®tzl et al., 1995). The mature form of murine PrP C consists of 209 amino acids (Westaway et al., 1987) (corresponding to residues 23 to 231 in hamster PrP) and appears to be necessary for normal synaptic function (Collinge et al., 1994) and long-term survival of Purkinje neurons (Sakaguchi et al., 1996). PrP C has a single disulfide bond between residues 179 and 214, two N-glycosylation sites at residues 181 and 197 and is attached to the cellular surface via a glycosyl phosphatidyl inositol (GPI) anchor at the carboxy-terminal serine 231 (Stahl & Prusiner, 1991)
To study folding of the predicted four-helix bundle domain 108-218 of PrP C , we recombinantly expressed two segments of mouse PrP comprising residues 95 to 231 and 107 to 231 in Escherichia coli. Both segments were fused to the bacterial OmpA sequence for secretory periplasmic expression to allow formation of the single disulfide bond. Expression yielded large amounts of soluble protein in the periplasmic fraction. However, Edman sequencing revealed that the segments 95-231 and 107-231 were amino-terminally degraded in vivo. All cleavage sites were found within the predicted first helix of PrP C (amino acids 109 to 122) after residues 112, 118, and 120.
The data were evaluated by a six-parameter fit and normalized as described (Santoro & Bolen, 1988).intrinsically stable folding unit PrP(121 to 231) is not in accordance with the proposed three-dimensional structure of the PrP C segment 108-218 (Huang et al., 1994) as it lacks the first helix (residues 109 to 122) of the predicted four-helix bundle. However, several lines of evidence suggest that the main and possibly the only part of PrP C with defined three-dimensional structure is represented by residues 121 to 231, and that most of the proposed structural changes linked to the transition from PrP C to PrP Sc may occur within this segment:
(1) Amino- or carboxy-terminal secondary structure elements being part of a single, small protein domain generally cannot be deleted without loss of protein stability and cooperativity of folding, since one-domain modules appear to fold in a concerted, cooperative mechanism and not in a hierarchical process (de Prat Gay et al., 1995a,b).
(2) It was demonstrated that peptide fragments comprising isolated helices of myohemerythrin, a four-helix bundle protein, spontaneously form helical structures (Dyson et al., 1992). However, a synthetic peptide spanning the first, predicted a-helix (residues 106 to 126) did not exhibit a-helical structure in aqueous solution and appeared to be a mixture of b-sheet and random coil structure (de Gioia et al., 1994).
(3) Proteolytic cleavage of a protein domain should occur mainly at exposed loop regions rather than within secondary structure elements (Price & Johnson, 1993). The experimental evidence thus indicates that the fold of PrP(121-231) is incompatible with the structure previously proposed for PrP C . It was recently shown that transgenic mice exclusively expressing a PrP variant lacking residues 32 to 80 are still susceptible to infection by mouse PrP Sc and capable of propagating the infectious agent, which means that all residues required for the conversion to PrP Sc are located within segment 81-231 (Fischer et al., 1996). PrP(121-231) represents 74% of this segment and contains ten of the 13 codons in human PrP, where point mutations are assumed to be associated with inherited prion diseases (Scha®tzl et al., 1995; Prusiner, 1993). The polymorphism at residue 129 in human PrP, which appears to influence susceptibility to the Creutzfeldt-Jakob disease (Palmer et al., 1991), also lies within PrP(121-231). Therefore, it will be most interesting to see whether transgenic mice exclusively expressing the autonomously folding fragment PrP(121-231) will still be susceptible to scrapie.
The reversibility of PrP(121-231) folding raises principle questions on the folding pathways of PrP C and PrP Sc . When we neglect an autocatalytic mechanism for formation of monomeric PrP Sc and simply assume that formation of insoluble, protease-resistant PrP Sc -oligomers ((PrP Sc )n )is an irreversible process an oligomerization-competent PrP Sc monomer may be: (1) by an on-pathway or (2) by an off-pathway folding intermediate of PrP C (both of which could be molten globule-like; Safar et al., 1994); or (3) the final product of PrP folding, protein and difference in accessible surface area between the unfolded and folded state (Myers et al., 1995), has a value of 8.6 (20.5) kJ mol -1 M -1 GdmCl and is in the range expected for a 13.3 kDa protein (Myers et al., 1995). We have shown that the amino-terminally truncated segment of the mouse prion protein (residues 121 to 231) is an isolated domain with tertiary structure and high intrinsic stability, whose folding and solubility does not require N-glycosylation at residues 181 and 198. Most importantly, refolding of chemically denatured PrP(121-231) is cooperative and reversible and yields a molecule indistinguishable from the native recombinant protein which would mean that PrP C is a kinetically trapped folding intermediate of PrP Sc .
All models predict that the kinetics of formation of PrP Sc -oligomers are strongly dependent on protein concentration, which is consistent with the observation that transgenic mice overexpressing the natural prion protein exhibit a strong increase in susceptibility to infection by prions (Bu® eler et al., 1993; Fischer et al., 1996). Since examples for protein folding reactions under kinetic control are extremely rare and have so far only been reported for polypeptides with pro-sequences (Baker et al., 1992; Eder et al., 1993a,b), model (3) appears to be most unlikely, and the conformation of recombinant PrP(121-231) may indeed be identical with its three-dimensional fold in the natural PrP C protein. We believe that the high stability and solubility of PrP(121-231) over a wide range of conditions provides the basis to investigate its kinetics of folding and unfolding, to identify the nature of possible folding intermediates of PrP experimen-tally (Baldwin, 1996), and to solve the three-dimensional structure of the folded part of PrP C , which is of central importance for understanding the molecular mechanisms underlying the trans-mission of prion diseases.
Baker, D., Sohl, J. L. & Agard, D. A. (1992). A protein-folding reaction under kinetic control. Nature, 356, 263‚265. Baldwin, R. L. (1996). On-pathway versus off-pathway folding intermediates. Folding & Design, 1, R1‚R8. Bu® eler, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M. & Weissmann, C. (1993). Mice devoid of PrP are resistant to scrapie. Cell, 73, 1339‚1347. Collinge, J., Whittington, M. A., Sidle, K. C., Smith, C. J., Palmer, M. S., Clarke, A. R. & Jefferys, J. G. R. (1994). Prion protein is necessary for normal synaptic function. Nature, 370, 295‚297. de Gioia, L., Selvaggini, C., Ghibaudi, E., Diomede, L., Bugiani, G. F., Forloni, G., Tagliavini, F. & Salmona, M. (1994). Conformational polymorphism of the amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein. J. Biol. Chem. 269, 7859‚7862. de Prat Gay, G., Ruiz-Sanz, J., Neira, J., Itzhaki, L. S. & Fersht, A. R. (1995a). Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module. Proc. Natl Acad. Sci. USA, 92, 3683‚3686. de Prat Gay, G., Ruiz-Sanz, J., Neira, J., Corrales, F. J., Otzen, D. E., Ladurner, A. G. & Fersht, A. R. (1995b). Conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its N terminus in vitro. Parallels with the protein folding pathway. J. Mol. Biol. 254, 968‚979. Dyson, H. J., Merutka, G., Waltho, J. P., Lerner, R. A. & Wright, P. E. (1992). Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myo-hemerythrin. J. Mol. Biol. 226, 795‚817. Eder, J., Rheinnecker, M. & Fersht, A. R. (1993a). Folding of subtilisin BPN': characterization of a folding intermediate. Biochemistry, 32, 18‚26. Eder, J., Rheinnecker, M. & Fersht, A. R. (1993b). Fold-ing of subtilisin BPN': role of the pro-sequence. Biochemistry, 32, 18‚26. Fischer, M., Ru® licke, T., Raeber, A., Sailer, A., Moser, M., Oesch, B., Brandner, S., Aguzzi, A. & Weissmann, C. (1996). Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J. 15, 1255‚1264. Gill, S. C. & von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319‚326. Griffith, J. S. (1967). Self-replication and scrapie. Nature, 215, 1043‚1044. Huang, Z., Gabriel, J.-M., Baldwin, M. A., Fletterick, R. J., Prusiner, S. B. & Cohen, F. E. (1994). Proposed three-dimensional structure for the cellular prion protein. Proc. Natl Acad. Sci. USA, 91, 7139‚7143. Huang, Z., Prusiner, S. B. & Cohen, F. E. (1995). Scrapie prions: a three-dimensional model of an infectious fragment. Folding Design, 1, 13‚19. Johnson, W. C., Jr (1990). Protein secondary structure and circular dichroism: practical guide. Proteins: Struct. Funct. Genet. 7, 205‚214. Kazmirski, S. L., Alonso, D. O. V., Cohen, F. E., Prusiner, S. B. & Daggett, V. (1995). Theoretical studies of the sequence effects on the conformational properties of a fragment of the prion protein: implications for scrapie formation. Chem. Biol. 2, 305‚315. Myers, J. K., Pace, C. N. & Scholtz, J. M. (1995). Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138‚2148. Pace, C. N. (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131, 266‚280. Reversible Folding of N-terminally Truncated PrP 619 Palmer, M. S., Dryden, A. J., Hughes, J. T. & Collinge, J. (1991). Homozygous prion protein genotype predis-poses to sporadic Creutzfeldt-Jakob disease. Nature, 352, 340‚342. Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E. & Prusiner, S. B. (1993). Conversion of a-helices into b-sheets features in the formation of the scrapie prion proteins. Proc. Natl Acad. Sci. USA, 90, 10962‚10966. Price, N. C. & Johnson, C. M. (1993). Proteinases as probes of conformation of soluble proteins. In Proteolytic Enzymes, A Practical Approach (Beynon, R. J. & Bond, J. S., eds), pp. 163‚180, IRL Press, Oxford. Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. Science, 216, 136‚144. Prusiner, S. B. (1991). Molecular biology of prion diseases. Science, 252, 1515‚1522. Prusiner, S. B. (1993). Genetic and infectious prion diseases. Arch. Neurol. 50, 1129‚1153. Safar, J., Roller, P. P., Gajdusek, D. C. & Gibbs, C. J., Jr (1994). Scrapie amyloid (prion) protein has the conformational characteristics of an aggregated molten globule folding intermediate. Biochemistry, 33, 8375‚8383. Santoro, M. M. & Bolen, D. W. (1988). Unfolding free energy changes determined by the linear extrapol-ation method. 1. Unfolding of phenylmethanesul-fonyl a-chymotrypsin using different denaturants. Biochemistry, 27, 8063‚8068. Sakaguchi, S., Katamine, S., Nishida, N., Moriuchi, R., Shigematsu, K., Sugimoto, T., Nakatani, A., Katoaka, Y., Houtani, T., Shirabe, S., Okada, H., Hasegawa, S., Miyamoto, T. & Noda, T. (1996). Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature, 380, 528‚531. Scha®tzl, H. M., Da Costa, M., Taylor, L., Cohen, F. E. & Prusiner, S. B. (1995). Prion protein gene variation among primates. J. Mol. Biol. 245, 362‚374. Stahl, N. & Prusiner, S. B. (1991). Prions and prion proteins. FASEB J. 5, 2799‚2807. Strobl, S., Mu® hlhahn, P., Bernstein, R., Wiltscheck, R., Maskos, K., Wunderlich, M., Huber, R., Glockshuber, R. & Holak, T. A. (1995). Determination of the three-dimensional structure of the bifunctional a-amylase/trypsin inhibitor from Ragi seeds by NMR-spectroscopy. Biochemistry, 34, 8281‚8293. Studier, F. W. & Moffatt, B. A. (1986). Use of bac-teriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113‚130. Weissmann, C. (1994). Molecular biology of prion diseases. Trends Cell Biol. 4, 10‚14. Weissmann, C. (1995). Yielding under the strain. Nature, 375, 620‚628. Westaway, D., Goodman, P. A., Mirenda, C. A., McKinely, M. P., Carlson, G. A. & Prusiner, S. B. (1987). Distinct prion proteins in short and long scrapie incubation period mice. Cell, 51, 651‚662.