Kinetic Intermediate in the Folding of Human Prion Protein

Abstract

Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90–231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrPSc isoform. Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90–231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrPSc isoform. Transmissible spongiform encephalopathies, or prion diseases, are a group of intriguing neurodegenerative disorders that include scrapie in sheep and goat, bovine spongiform encephalopathy in cattle, chronic waste disorder in deer and elk, and Creutzfeldt-Jacob disease in humans (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). These diseases are associated with conformational conversion of a normal prion protein, PrPC, 1The abbreviations used are: PrPC, cellular prion protein; PrP, prion protein; PrPSc, pathogenic PrP isoform.1The abbreviations used are: PrPC, cellular prion protein; PrP, prion protein; PrPSc, pathogenic PrP isoform. into a misfolded isoform, PrPSc. According to the “protein-only” hypothesis (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar), the transmission of the disease does not require nucleic acids, and the prion pathogen consists solely of PrPSc. The latter conformer is believed to act as an infectious agent by catalyzing self-propagating conversion of endogenous PrPC into the pathogenic PrPScisoform. Although the ultimate proof for the protein-only hypothesis is still missing (2Caughey B. Nat. Med. 2000; 6: 751-754Google Scholar), the central role of prion protein in the pathogenesis of the disease is documented by a wealth of biochemical and genetic data (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). Cellular human prion protein, PrPC, is a glycoprotein that contains a disulfide bond, is N-glycosylated, and is attached to the plasma membrane by a glycosyl phosphatidylinositol anchor (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). NMR studies have shown that the recombinant prion protein in solution consists of a largely unordered N-terminal region and the folded C-terminal domain encompassing three α-helices and a short β-sheet (3Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wuthrich K. Nature. 1996; 382: 180-182Google Scholar, 4Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Google Scholar, 5Zahn R. Liu A. Luhrs T. Riek R. Von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Google Scholar). Recent crystallographic studies have captured the C-terminal part of PrP as a domain-swapped dimer (6Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Google Scholar). This dimer, which is only marginally populated in solution and selectively crystallizes, is also α-helical, and its overall fold is similar to that of the monomer. The PrPC → PrPSc conversion, which appears to occur without any covalent modifications, is believed to involve a major conformational change in the prion protein (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). In contrast to a largely α-helical PrPC, the pathogenic PrPSc isoform is characterized by a high content of β-sheet structure (7Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Google Scholar, 8Gasset M. Baldwin M.A. Fletterick R.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Google Scholar), partial resistance to proteolytic digestion, and a propensity to aggregate into insoluble amyloid-like fibrils and plaques (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). Despite intensive research, the molecular mechanism underlying the PrPC → PrPScconversion and prion propagation remains enigmatic. The key to understanding this mechanism is to elucidate the folding pathway(s) of the prion protein. Here we present evidence for a population of a kinetic intermediate during the folding of the human prion protein. This species may represent a crucial monomeric precursor on the pathway of prion protein conversion to the pathogenic PrPScisoform. The plasmid encoding huPrP-(90–231) with an N-terminal linker containing a His6 tail and a thrombin cleavage site was described previously (9Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Google Scholar, 10Morillas M. Vanik D.L. Surewicz W.K. Biochemistry. 2001; 40: 6982-6987Google Scholar). The Y218W and F175W variants were constructed by site-directed mutagenesis using appropriate primers and the QuikChange kit (Stratagene). The proteins were expressed, cleaved with thrombin, and purified according to the previously described protocol (9Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Google Scholar). The equilibrium unfolding curves were obtained using a Jasco J-810 spectropolarimeter equipped with an automated titrator and a temperature control system. In these experiments, native protein (1.4 μm) in an appropriate buffer was titrated with a 9 m buffered urea solution containing protein at the same concentration. The extent of protein unfolding was monitored by ellipticity at 222 nm or fluorescence intensity above 320 nm (excitation wavelength of 296 nm). The unfolding curves were analyzed using a two-state transition model (11Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Google Scholar). The kinetics of unfolding and refolding reactions were studied by diluting the native protein or protein fully unfolded in 8 m urea into the buffer (50 mm phosphate, pH 7.0 or 50 mm sodium acetate, pH 4.8) containing urea at a desired concentration. In most experiments, the final protein concentration was 5.0 μm. The reactions were monitored by fluorescence above 320 nm (excitation at 296 nm) using the Applied Photophysics π* stopped-flow instrument equipped with a 5-μl cell and operating at a 1:10 mixing ratio. Depending on urea concentration, the mixing dead time of this instrument was 0.8–1.1 ms. The dead times were determined by a standard protocol for the reduction of 2,6-dichlorophenolindophenol byl-ascorbate (12Tonomura B. Nakatani H. Ohnishi M. Yamaguchi-Ito J. Hiromi K. Anal. Biochem. 1978; 84: 370-383Google Scholar). Each protein folding/unfolding reaction was measured at least eight times. The kinetic traces were averaged and analyzed using the software provided by Applied Photophysics. In the present study, we have focused on folding of the recombinant protein corresponding to human PrP fragment 90–231 (huPrP-(90–231)). This region of PrP is of special importance because it encompasses the entire sequence of proteinase-resistant protein found in prion-infected brain, contains all known point mutations associated with familial prion disorders, and is sufficient for the propagation of the disease. The folding mechanism of huPrP-(90–231) was studied by the kinetic stopped-flow method with tryptophan fluorescence detection. Since the fluorescence of the sole native Trp at position 99 changes very little upon protein unfolding, these studies required preparation of huPrP-(90–231) variants with a single Trp engineered into the folded domain of the protein. This was accomplished by replacing Trp99 with Phe and, on that background, substituting Phe or Tyr at different positions with tryptophan. Preliminary characterization of these single Trp variants has identified the Y218W mutant (Fig.1) as potentially the most suitable candidate for stopped-flow experiments. Both at neutral and acidic pH, the Y218W substitution is essentially non-perturbing as indicated by far-UV circular dichroism spectra and equilibrium urea unfolding curves (data not shown). Furthermore, the fluorescence of the Trp218 probe is very sensitive to protein conformation. It is quenched in the native state, increasing sharply upon huPrP-(90–231) unfolding in urea. The stopped-flow method was used to follow kinetics of Y218W huPrP-(90–231) folding and unfolding at both acidic and neutral pH. The experiments were performed at 5 °C because, as shown previously (13Wildegger G. Liemann S. Glockshuber R. Nat. Struct. Biol. 1999; 6: 550-553Google Scholar), at room temperature the refolding and unfolding reactions for the prion protein are very fast, occurring within the dead time of the stopped-flow instrument. Representative kinetic traces for the fluorescence-detected folding and unfolding reactions of Y218W huPrP-(90–231) at pH 4.8 are shown in Fig.2. At each denaturant concentration, the kinetic traces could be fitted by a single exponential function, yielding the apparent rate constants. The urea dependence of these rate constants (“chevron plot”) is shown in Fig.3 A. The data do not fit a two-state transition model since the plot of the logarithm of the refolding rate versus [denaturant] at low urea concentrations (below ∼3.5 m) clearly deviates from linearity. The unfolding branch of the curve does not show any detectable curvature; however, the urea concentration range available for unfolding measurements is too narrow to allow any definitive conclusions. A non-linearity of the refolding part of the chevron plot (below 3 m urea) was also observed at pH 7 (Fig.3 B). Similar downward curvatures of the rate constant versus [denaturant] plots have been reported for a number of other proteins (14Roder H. Colon W. Curr. Opin. Struct. Biol. 1997; 7: 15-28Google Scholar, 15Fersht A. Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman and Company, New York1998Google Scholar, 16Ferguson N. Capaldi A.P. James R. Kleanthous C. Radford S.E. J. Mol. Biol. 1999; 286: 1597-1608Google Scholar). This effect is usually attributed to the presence of an early folding intermediate that forms during the dead time of the instrument and becomes increasingly populated under stabilizing conditions (i.e. at low denaturant concentration) (14Roder H. Colon W. Curr. Opin. Struct. Biol. 1997; 7: 15-28Google Scholar, 15Fersht A. Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman and Company, New York1998Google Scholar, 16Ferguson N. Capaldi A.P. James R. Kleanthous C. Radford S.E. J. Mol. Biol. 1999; 286: 1597-1608Google Scholar). However, recent studies indicate that deviation from linearity in chevron plots could also result from the transient protein aggregation during the refolding reaction (17Silow M. Oliveberg M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6084-6086Google Scholar). To test the latter possibility, the measurements of refolding kinetics at a few urea concentrations (1.2, 1.8, 2.3, and 2.9 m) were repeated using protein concentration of 1.2, 2.7, 5.0, and 25 μm. The rate constants did not show any appreciable protein concentration dependence, indicating that, under the conditions used, the folding reaction is monomolecular and does not involve any transient aggregates.Figure 3Folding and unfolding data for Y218W huPrP-(90–231) at pH 4.8 (A and C) and pH 7 (B and D). Aand B, rates of refolding (low denaturant concentrations) and unfolding (high denaturant concentrations) on the log scaleversus urea concentration. C and D, initial fluorescence intensities extrapolated to time 0 for the refolding (▿) and unfolding (▵) reactions together with the equilibrium fluorescence intensities at long times (●). The concentration of huPrP-(90–231) was 5.0 μm. Thedashed lines are linear extrapolations of the base lines for the native and fully unfolded states. The solid linesrepresent the best fit of experimental data to a sequential three-state kinetic model N ↔ I ↔ U.View Large Image Figure ViewerDownload (PPT) The presence of a kinetic folding intermediate at pH 4.8 was further corroborated by the analysis of the refolding amplitudes. At low urea concentrations, the initial fluorescence intensities obtained by extrapolation of the kinetic curves to time 0 are markedly smaller than the amplitudes expected assuming that the fluorescence intensity of the unfolded state changes linearly with denaturant concentration (Fig.3 C). This indicates that a significant fraction of the total fluorescence change occurs within the dead time of the instrument (burst phase), suggesting rapid formation of a folding intermediate (14Roder H. Colon W. Curr. Opin. Struct. Biol. 1997; 7: 15-28Google Scholar, 15Fersht A. Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman and Company, New York1998Google Scholar, 16Ferguson N. Capaldi A.P. James R. Kleanthous C. Radford S.E. J. Mol. Biol. 1999; 286: 1597-1608Google Scholar). At pH 7, the unfolding curve was shifted to very high urea concentrations. The unfolding base line for Y218W huPrP-(90–231) under these conditions was poorly defined (Fig. 3 D), precluding application of the “amplitude test.” A non-linearity of chevron plots and the observation of a burst phase strongly indicate the presence of at least one intermediate on the Y218W huPrP-(90–231) folding pathway. It has been that chevron plots could also result from changes in the transition state M. Oliveberg M. Biochemistry. 1999; Scholar). However, this is to be a significant in the present study, since transition state could not for the observed burst phase in the refolding the denaturant dependence of the folding rates at both pH studied (Fig. to the folding reaction according to the three-state I and N represent the unfolded and native and I represents an early folding intermediate during the dead time of the instrument N. Capaldi A.P. James R. Kleanthous C. Radford S.E. J. Mol. Biol. 1999; 286: 1597-1608Google Scholar). The indicate that, to the unfolded state, the intermediate is under acidic conditions than at neutral pH of and at pH 7 and In the intermediate to the native state at a rate of The fit also the of the for the to the m m m The which a of the of that becomes upon formation of I (14Roder H. Colon W. Curr. Opin. Struct. Biol. 1997; 7: 15-28Google Scholar), indicates that the folding intermediate is (i.e. characterized by a smaller under acidic conditions than at neutral and kinetic for the folding of human prion represents the obtained from the best fit of the equilibrium unfolding data to a two-state model (11Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Google Scholar). The are from the best fit of data of 3 and to a three-state sequential model as shown in I according to the N. Capaldi A.P. James R. Kleanthous C. Radford S.E. J. Mol. Biol. 1999; 286: 1597-1608Google is the observed rate and are rate constants corresponding to the I → N and N → I and is the equilibrium constant the unfolded and intermediate states. The m and represent the denaturant concentration dependence of and was from kinetic data according to the and The to the of the I state, was as in a represents the obtained from the best fit of the equilibrium unfolding data to a two-state model (11Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Google Scholar). The are from the best fit of data of 3 and to a three-state sequential model as shown in I according to the N. Capaldi A.P. James R. Kleanthous C. Radford S.E. J. Mol. Biol. 1999; 286: 1597-1608Google is the observed rate and are rate constants corresponding to the I → N and N → I and is the equilibrium constant the unfolded and intermediate states. The m and represent the denaturant concentration dependence of and was from kinetic data according to the and The to the of the I state, was as The present are at with previously reported for prion protein domain on data for the F175W of it was that at neutral pH prion protein according to the two-state mechanism without any kinetic (13Wildegger G. Liemann S. Glockshuber R. Nat. Struct. Biol. 1999; 6: 550-553Google Scholar). In of this apparent we have repeated measurements at pH 7 for F175W using the same group as in the with prion protein. The rate for F175W huPrP-(90–231) was very similar to that of the Y218W a defined curvature at low denaturant concentrations Furthermore, a clear from a linear base line was observed for initial fluorescence intensities indicating the presence of a burst phase in the refolding reaction (Fig. B). The fit of kinetic data according to a three-state model for F175W folding very similar to for the Y218W The of the obtained using the in different of the protein 3 and in Y218W and F175W (Fig. indicates that the single Trp variants used in this represent the folding of the native human prion protein. A number of could to the the present data and the apparent of a folding intermediate in the with prion protein. It is that the human and PrP fold different that the N-terminal fragment the folding of the C-terminal domain with PrP were performed using the the present with human protein used a 90–231 However, it also be that data reported for refolding are to a narrow urea concentration range above The missing data 3 m urea are of potentially importance since folding curvatures in chevron usually detectable only under stabilizing at low denaturant concentrations (14Roder H. Colon W. Curr. Opin. Struct. Biol. 1997; 7: 15-28Google Scholar, 15Fersht A. Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman and Company, New York1998Google Scholar). at high urea concentrations the rate constants for huPrP-(90–231) refolding at pH 7 are very similar to reported for and is a linear dependence of the logarithm of these rates on denaturant concentration. A from this of a folding becomes detectable only at urea concentrations 3 at low urea concentrations in the present to the recent in stopped-flow The of a 5-μl cell the dead time of instrument to measurements of rate constants in of In a recent with it has been that the stopped-flow measurements of reactions that are to the dead time may be to experimental Biochemistry. 2000; Scholar). However, this to folding reactions in which the detectable phase is by poorly The present studies with prion protein be largely to these since the observed kinetic traces are clearly the of the protein-only the key in the pathogenesis of prion diseases is the conversion of prion protein from α-helical form, PrPC, to a PrPSc (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). of the central to the mechanism of this conversion to the of the PrP that is a precursor of the oligomeric PrPSc. it a native α-helical a fully unfolded form, or a monomeric folding point to a in the PrPC → PrPScconversion of folding F.E. Baldwin M. Fletterick R.J. Prusiner S.B. Science. Scholar). intermediate are also believed to a central role in formation by other However, for proteins as and variants 1997; M. P.E. Radford S.E. Nature. 1997; Scholar), folding for prion protein have very to and (13Wildegger G. Liemann S. Glockshuber R. Nat. Struct. Biol. 1999; 6: 550-553Google Scholar, A. J. Nat. Struct. Biol. 1999; 6: Scholar, H. Prusiner S.B. Cohen F.E. S. J. Mol. Biol. Scholar). studies with the recombinant prion protein have identified an of PrP that was to represent a equilibrium W. R. Gambetti P. Surewicz W.K. J. Biol. Chem. 1997; Scholar, S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: Scholar). However, recent data have this that the species is not an equilibrium folding intermediate in represents an of prion protein with similar to of PrPSc M. Vanik D.L. Surewicz W.K. Biochemistry. 2001; 40: 6982-6987Google Scholar, W. Morillas M. Gambetti P. Surewicz W.K. Biochemistry. 2000; Scholar). the the of in prion protein folding remains a of The present kinetic stopped-flow for the experimental evidence that prion protein by a three-state mechanism a monomeric This early which the formation of the native state, appears to be and under acidic In addition to its role in normal prion protein folding, the folding intermediate is also likely to be of major importance in the PrPC → In the of experimental evidence for an have that the mechanism of prion protein conversion is different from that for other proteins and that it is the unfolded state of PrP that is into PrPSc (13Wildegger G. Liemann S. Glockshuber R. Nat. Struct. Biol. 1999; 6: 550-553Google Scholar, A. J. Nat. Struct. Biol. 1999; 6: Scholar). However, the present of a monomeric intermediate in prion protein folding for a of this with the fully unfolded protein, the PrP intermediate is with a high and aggregation propensity of folding the I state of PrP as a candidate than the unfolded state for a monomeric precursor of PrPSc. The role of a folding intermediate in the PrP conversion is in line with the recent that the transition of the recombinant prion protein to a is strongly in the presence of low concentrations of under conditions that the population of an intermediate state M. Vanik D.L. Surewicz W.K. Biochemistry. 2001; 40: 6982-6987Google Scholar). In conditions the native state or that equilibrium the fully unfolded state (high concentration of are not to the conversion reaction M. Vanik D.L. Surewicz W.K. Biochemistry. 2001; 40: 6982-6987Google Scholar). It also be that the in transition of the recombinant PrP to a appears to be at low pH W. Morillas M. Gambetti P. Surewicz W.K. Biochemistry. 2000; Scholar, A. J. H. J. Science. 1999; Scholar). This with the present observation that acidic pH the intermediate populated during the prion protein folding acidic conditions are of to prion disease pathogenesis since indicate that the PrPC → PrPSc conversion may in acidic B. Ernst D. J. 1991; Scholar). The evidence of an intermediate in prion protein folding also has potentially for understanding the mechanism of familial diseases that with mutations in human prion protein (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). In these disorders, the PrPC → PrPSc conversion appears to occur not with a prion It has been that familial mutations the conversion of the prion protein by the native structure of PrPC F.E. Baldwin M. Fletterick R.J. Prusiner S.B. Science. Scholar). However, recent data indicate that of these mutations of the native state of prion protein to the unfolded state W. S.B. Gambetti P. Surewicz W.K. J. Biol. Chem. 1998; Scholar, S. Glockshuber R. Biochemistry. 1999; Scholar), a for the model assuming that a fully unfolded prion protein is a precursor of PrPSc. The of an intermediate in prion protein folding may this on the present we that familial mutations the conversion reaction not by increasing the population of an unfolded protein by the increasing the of the folded intermediate This a for the effect of mutations on the folding pathway of the prion protein. are to W. Swietnicki for the to the molecular part of this