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• W2149247478 abstract "Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrPC, into a pathogenic conformer, PrPSc. Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrPC → PrPSc conversion in the E200K form of hereditary prion disease. Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrPC, into a pathogenic conformer, PrPSc. Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrPC → PrPSc conversion in the E200K form of hereditary prion disease. Creutzfeldt-Jakob disease Gerstmann-Sträussler-Scheinker disease fatal familial insomnia prion protein cellular PrP isoform scrapie (proteinase-resistant) PrP isoform human heteronuclear single-quantum coherence nuclear Overhauser effect nuclear Overhauser effect spectroscopy two dimensional, 3D, three-dimensional atomic root mean square Spongiform encephalopathies, or prion diseases, are a novel class of neurodegenerative disorders that affect animals and humans. They include scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease (CJD),1Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru in humans. These diseases may arise sporadically, may be inherited, or may be acquired by transmission of an infectious agent (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar, 2Horiuchi M. Caughey B. Structure. 1999; 7: R231-R240Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). According to the “protein only” hypothesis (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar, 2Horiuchi M. Caughey B. Structure. 1999; 7: R231-R240Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 3Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4097) Google Scholar), the key event in the pathogenesis of prion disorders is the conversion of a normal prion protein, PrPC, into a pathogenic (scrapie) form, PrPSc. The latter protein accumulates in the diseased brain and is believed to be the sole component of the infectious prion pathogen. The transition between PrPC and PrPScoccurs post-translationally without any detectable covalent modifications to the protein molecule (4Stahl N. Baldwin M.A. Teplow D.B. Hood L. Gibson B.W. Burlingame A.L. Prusiner S.B. Biochemistry. 1993; 32: 1991-2002Crossref PubMed Scopus (536) Google Scholar). The two protein isoforms have profoundly different physicochemical properties. PrPCis highly soluble and easily degraded by proteinase K, whereas PrPSc exists as an insoluble aggregate that is resistant to proteinase K digestion and often has the characteristics of an amyloid (5Meyer R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (517) Google Scholar, 6Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1251) Google Scholar). These differences in physical properties most likely reflect different conformations of the two isoforms. Indeed, spectroscopic data show that PrPC is highly α-helical, whereas PrPSc contains a large proportion of β-sheet structure (7Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusinec S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2075) Google Scholar, 8Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar, 9Safar J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. Protein Sci. 1993; 2: 2206-2216Crossref PubMed Scopus (174) Google Scholar, 10Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wuthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1128) Google Scholar). In addition to biochemical evidence, the protein only hypothesis is supported by experiments showing the resistance of PrP knockout mice to the infectious scrapie agent (11Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1810) Google Scholar). Another argument in favor of this hypothesis is the link between hereditary human spongiform encephalopathies (i.e. familial CJD, GSS, and FFI) and specific mutations in the gene coding for the human prion protein (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar,12Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4611-4614Crossref PubMed Scopus (65) Google Scholar). A key to understanding the mechanism by which familial mutations facilitate the pathogenic process is to determine how these mutations affect the conformational structure of prion protein. In the present study, we report the three-dimensional structure of the folded domain of the recombinant human prion protein containing a mutation (E200K) corresponding to the most common familial form of Creutzfeldt-Jakob disease. The E200K mutant was constructed using the QuikChange™ kit (Stratagene, La Jolla, CA) and primers 5′-GAACTTCACCAAGACCGACGTTAAG-3′ and 5′-CTTAACGTCGGTCTTGGTGAAGTTC-3′. The template DNA was the pRSETB vector with the cDNA corresponding to the wild-type huPrP-(90–231) inserted into the multiple cloning site as described previously (13Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The protein was expressed, refolded, and purified using the procedures described previously (13Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). To obtain15N-/13C-labeled protein, bacteria were grown in a minimum medium containing 15NH4Cl (1 g/l) and 13C6-glucose (2 g/l) as a sole source of nitrogen and carbon, respectively. Samples for NMR spectroscopy were prepared at a protein concentration of 0.7–0.9 mm in 10 mm d 4-sodium acetate buffer containing 0.005% sodium azide and 10% (v/v) D2O, pH 4.6. Most NMR measurements were performed at 26 °C on a Varian INOVA 600 spectrometer equipped with a triple-resonance gradient probe. Some15N-HSQC, HCCH-total correlation spectroscopy, 100-ms 13C-NOESY, and 15N-NOESY spectra were also acquired on a Bruker Avance 800-MHz spectrometer. All 3D spectra were processed by NMRPIPE (14Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11536) Google Scholar) and analyzed with PIPP (15Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). 2D spectra were processed and analyzed with the program FELIX 98 (Molecular Simulations, Inc.). Nearly complete backbone 1H, 13C, and 15N NMR assignments were achieved using standard heteronuclear methodology (16Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (791) Google Scholar) with the assistance of AUTOASSIGN (17Zimmerman D.E. Kulikowski C.A. Huang Y. Feng W. Tashiro M. Shimotakahara S. Chien C. Powers R. Montelione G.T. J. Mol. Biol. 1997; 269: 592-610Crossref PubMed Scopus (265) Google Scholar). Slowly exchanging amide protons were identified by dissolving the lyophilized protein in D2O and collecting 15N-HSQC spectra as a function of time. The NOE distance restraints were determined from 3D15N-edited NOESY (100 ms),15N-/13C-edited NOESY (100 ms) in H2O, 13C-edited NOESY (50 and 100 ms) in D2O, and 2D NOESY spectra (100 ms).JHNHA coupling constants were measured from 3D HNHA spectra. Backbone dihedral angle restraints were added on the basis of the chemical shift indices for the Hα, Cα, and carbonyl resonances. Stereospecific assignments were obtained by direct analysis of HNHB data and short mixing time13C-NOESY (50 ms) experiments. Structure calculations were performed with the programs ARIA (18Nilges M. Macias M.J. O'Donoghue S.I. Oschkinat H. J. Mol. Biol. 1997; 269: 408-422Crossref PubMed Scopus (388) Google Scholar)/CNS (19Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) using the standard protocol, except that the upper limit for methyl groups was increased by 0.2 Å. Hydrogen-deuterium exchange rates were measured at 26 °C, and the protection factors were calculated as described previously (20Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins. 1993; 17: 75-86Crossref PubMed Scopus (1755) Google Scholar). The structure of the E200K variant of huPrP-(90–231) was characterized by NMR spectroscopy. In close similarity to the wild-type human (21Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar), mouse (22Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (664) Google Scholar), and Syrian hamster prion protein (23Liu H. Farr-Jones S. Ulyanov N.B. Llinas M. Marqusee S. Groth D. Cohen F.E. Prusiner S.B. James T.L. Biochemistry. 1999; 38: 5362-5377Crossref PubMed Scopus (197) Google Scholar, 24Donne 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: 13452-13457Crossref PubMed Scopus (637) Google Scholar), the N-terminal region (residues 90–124) of E200K huPrP-(90–231) is highly flexible. Present data provides no evidence of any long range interactions or local structural preferences within this part of the protein molecule. It should be noted that signal duplication observed previously for residues 120–122 in the Syrian hamster protein (23Liu H. Farr-Jones S. Ulyanov N.B. Llinas M. Marqusee S. Groth D. Cohen F.E. Prusiner S.B. James T.L. Biochemistry. 1999; 38: 5362-5377Crossref PubMed Scopus (197) Google Scholar) was not found for E200K huPrP-(90–231). Multiple signals were observed for several other residues in the flexible tail, but these signals likely result from cis forms of three Xxx-Pro peptide bonds present in this region (Fig. 1). The solution structure was calculated for the C-terminal domain 125–231 of the mutant protein. This domain consists of three α-helices that encompass residues 144–153 (helix 1), 172–194 (helix 2), and 200–227 (helix 3) and a short β-sheet formed by two strands at residues 129–131 and 161–163 (Fig. 2). A hydrogen bond between the amide proton of Met134 and the carbonyl oxygen of Asn159 suggests the presence of an irregular, β-bulge-type elongation of the second strand.Figure 2The three-dimensional structure of the folded domain (residues 125–228) in E200K huPrP. a, the ensemble of 30 energy-minimized conformers. b, Richardson representation of a representative structure (<SAr>). The α-helices are coloredred on the outside and yellow on the inside, whereas the β-strands are shown in cyan. The structures were displayed using the programs Insight II and MOLMOL (33Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6487) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The high quality of the present structural data is evident from statistical and PROCHECK analysis (25Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4405) Google Scholar) (TableI). Protein regions that encompass regular secondary structure elements display very high definition (r.m.s. deviations <0.4 Å). Loops are also well defined with r.m.s. deviations of 0.4–0.7 Å. The only undefined region in the folded domain is the loop connecting the second β-strand and helix 2 (residues 167–171). This loop is characterized by the absence of long- and medium-range NOEs. Further, the fact that some resonances in this region (such as the amide protons of Tyr169 and Ser170) could not be observed suggests the presence of multiple conformations, similar to the behavior of this region in the wild-type human prion protein (21Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar).Table IStructural statistics for E200K huPrP 90–231<SA> 1-a<SA> represents the 30 best structures of 60 calculated structures; <SAr> indicates the structure with lowest energy. The structures were calculated with ARIA/CNS (18, 19) using energy terms for NOE distance restraints, dihedral angle restraints, bonds, angles, impropers, and hard sphere Van der Waals radii.<SAr>Experimentally derived restraints 1-bNo distance restraints or dihedral angle restraints were violated by more than 0.2 Å or 3°, respectively. distance restraints intra (‖i-j‖ = 0)1070 sequential (‖i-j‖ = 1) 491 medium range (5 > ‖i-j‖ > 1) 514 long range (‖i-j‖ > 4) 579 unambiguous total2654 ambiguous total 516 3J HNHA-coupling constants 44 H bond restraints 1-c39 unambiguous and 3 ambiguous H bonds, two restraints per residue. 84 dihedral angle restraints 1-d67 phi, 67 psi, and 43 chi1. 177 Mean r.m.s. deviations from experimental distance (NOE) restraints (Å)0.0060 ± 0.00120.0040 dihedral restraints (degrees)0.2908 ± 0.05080.2191 J-coupling restraints (Hz)0.3438 ± 0.07090.2519 Mean r.m.s. deviations from idealized bond geometry (Å)0.0013 ± 0.000070.0012 angle geometry (degrees)0.2874 ± 0.00610.2744 improper geometry (degrees)0.2192 ± 0.00940.1952Energy (kcal/mol) total129.57 ± 7.40111.48 bonds2.97 ± 0.332.35 angles39.35 ± 1.6735.85 improper6.99 ± 0.605.54 Van der Waals 1-eA quartic repel potential was used in the calculations. Reported values were obtained with a final radius scaling constant of 1. Calculations were also performed with several other scaling factors between 0.78 and 1, without any noticeable effect on convergence or structural quality.67.89 ± 3.8459.06 NOE6.01 ± 2.432.61 dihedral0.94 ± 0.360.52Measures of structure quality 1-fThe overall quality of the structure was assessed using the program PROCHECK (25). Residues in most favorable region85.789.6 Residues in additional allowed region12.910.4 Residues in generously allowed regions 0.80 Residues in disallowed regions 0.60Coordinate precision 1-gr.m.s. deviations for the best 30 structures selected from the total ensemble of 60 calculated structures. (Å)125–231125–166; 171–228 backbone0.81 ± 0.140.45 ± 0.09 heavy1.17 ± 0.120.83 ± 0.091-a <SA> represents the 30 best structures of 60 calculated structures; <SAr> indicates the structure with lowest energy. The structures were calculated with ARIA/CNS (18Nilges M. Macias M.J. O'Donoghue S.I. Oschkinat H. J. Mol. Biol. 1997; 269: 408-422Crossref PubMed Scopus (388) Google Scholar, 19Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) using energy terms for NOE distance restraints, dihedral angle restraints, bonds, angles, impropers, and hard sphere Van der Waals radii.1-b No distance restraints or dihedral angle restraints were violated by more than 0.2 Å or 3°, respectively.1-c 39 unambiguous and 3 ambiguous H bonds, two restraints per residue.1-d 67 phi, 67 psi, and 43 chi1.1-e A quartic repel potential was used in the calculations. Reported values were obtained with a final radius scaling constant of 1. Calculations were also performed with several other scaling factors between 0.78 and 1, without any noticeable effect on convergence or structural quality.1-f The overall quality of the structure was assessed using the program PROCHECK (25Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4405) Google Scholar).1-g r.m.s. deviations for the best 30 structures selected from the total ensemble of 60 calculated structures. Open table in a new tab The amide exchange (proton to deuterium) experiments established that most of the residues located within regions of well defined secondary structure are solvent-shielded. The amide proton exchange protection factors for these residues are very similar to those reported for the wild-type human PrP (21Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar). However, some parts of helices show surprisingly little protection from the exchange. In particular, the C-terminal portions of helix 2 (residues 187–194) and helix 3 (residues 222–227) undergo essentially complete isotope exchange within less than 30 min, suggesting relatively large conformational flexibility in these regions. A similar finding was reported for the wild-type human PrP (21Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar). Overall, the 3D structure of the folded domain of E200K huPrP appears to be essentially identical to the structure previously reported for wild-type human prion protein (21Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar) (Fig.3). The boundaries between the secondary structure elements in these structures differ by one residue at most. The backbone r.m.s. differences are smaller than 2 Å for the entire folded domains and less than 1.5 Å for the non-loop regions. Slight differences seen among the structures (Fig. 3 a) are limited to loop 167–171, the first three residues in the loop connecting strand 1 and helix 1, and the C terminus of helix 2. However, factors such as flexibility and conformational averaging (see above) limit the accuracy of structural data for these regions in both the wild-type and mutant prion protein. Residue 200 is located at the beginning of helix 3 and is fully accessible to the solvent (Fig. 3 b). It is remarkable that the local structures of the wild-type and mutant protein in the vicinity of this residue are very similar. The only observable difference is the loss of salt-bridge interaction between the side chains of Glu200 and Lys204. In the wild-type protein, these side chains are intimately juxtaposed (within 5 Å) and therefore could be involved in a salt bridge (Fig. 3 c). In the mutant protein, the nearest negatively charged side chain to Lys200 is that of Asp196. However, these two side chains are 13 Å apart and thus too far for salt-bridge formation. Despite the essentially identical three-dimensional structure of the wild-type and mutant prion PrP, the Glu200 → Lys substitution has a major effect on the distribution of charges on the protein surface. At neutral pH values, the wild-type protein surface around Glu200 is characterized by largely negative electrostatic potential (Fig. 4). The E200K mutation breaks the relatively uniform distribution of charges, introducing patches of positive potential. The effect is even more dramatic under mildly acidic conditions (pH around 5) that lead to the protonation of His residues. Under such conditions, the surface around residue 200 in the wild-type protein contains patches of both negative and positive electrostatic potential, whereas the mutant protein surface is predominantly positively charged (Fig. 4). It is believed that transmissible spongiform encephalopathies are caused by a conversion of cellular prion protein, PrPC, into a conformationally altered form, PrPSc (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar, 2Horiuchi M. Caughey B. Structure. 1999; 7: R231-R240Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 3Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4097) Google Scholar). Of special interest are familial (hereditary) forms of the disease that are associated with specific mutations in the PrP gene. Because the PrPC → PrPSc conversion in hereditary diseases appears to occur spontaneously (i.e. not requiring infection with exogenous PrPSc), understanding how familial mutations affect the conformational structure and biophysical properties of prion protein should provide important clues regarding the molecular basis of the disease. It has been previously postulated that these mutations may facilitate the conversion reaction by destabilizing the native structure of PrPC (26Huang Z. Gabriel J.M. Baldwin M.A. Fletterick R.J. Prusiner S.B. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7139-7143Crossref PubMed Scopus (181) Google Scholar, 27Cohen F.E. Pan K.M. Huang Z. Baldwin M. Fletterick R.J. Prusiner S.B. Science. 1994; 264: 530-531Crossref PubMed Scopus (439) Google Scholar). However, recent biophysical studies showed that many of disease-associated mutations have essentially no effect on the thermodynamic stability of the recombinant PrP, suggesting that not all hereditary prion disorders can be rationalized through a common mechanism based on thermodynamic destabilization of the cellular prion protein (28Swietnicki W. Petersen R.B. Gambetti P. Surewicz W.K. J. Biol. Chem. 1998; 273: 31048-31052Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 29Liemann S. Glockshuber R. Biochemistry. 1999; 38: 3258-3267Crossref PubMed Scopus (295) Google Scholar). To gain further insight into the structural basis of hereditary transmissible spongiform encephalopathies, here we have determined the solution structure of the prion protein containing a Glu200 → Lys mutation that is associated with the familial form of CJD. Although Glu200 → Lys substitution has very little effect on the thermodynamic stability of prion protein (28Swietnicki W. Petersen R.B. Gambetti P. Surewicz W.K. J. Biol. Chem. 1998; 273: 31048-31052Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 29Liemann S. Glockshuber R. Biochemistry. 1999; 38: 3258-3267Crossref PubMed Scopus (295) Google Scholar), numerous observations argue for a causative role of this mutation in Creutzfeldt-Jakob disease. In particular, carriers of Glu200 → Lys mutation who live sufficiently long appear to invariably develop CJD and the resulting neuronal degeneration (30Chapman J. Ben-Israel J. Goldhammer Y. Korczyn A.D. Neurology. 1994; 44: 1683-1686Crossref PubMed Google Scholar). A striking result of the present study is the finding that the three-dimensional structure of the E200K variant is essentially identical to that previously reported for the wild-type prion protein. The only major consequence of the Glu200 → Lys substitution appears to be the redistribution of surface charges, resulting in a dramatically altered electrostatic potential in the mutant protein. The above surface-restricted changes are unlikely to affect the folding pattern or conformational transitions of an isolated protein molecule. Such changes could, however, have a profound effect on the ability of PrPC to interact with other molecules present in a complex cellular environment. In particular, mutation-dependent surface effects could facilitate the conversion reaction by inducing abnormal interactions of E200K PrPC with Protein X or other chaperones implicated in the pathogenesis of prion disorders (31Kaneko K. Zulianello L. Scott M. Cooper C.M. Wallace A.C. James T.L. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10069-10074Crossref PubMed Scopus (480) Google Scholar, 32DebBurman S.K. Raymond G.J. Caughey B. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13938-13943Crossref PubMed Scopus (229) Google Scholar). The Glu200 → Lys replacement could also contribute to the pathogenic process by interfering with normal membrane interactions of PrPC. The latter possibility is especially likely in view of recent data indicating that the stability and conformational transitions of prion protein are strongly affected by electrostatic interactions with cellular membranes (13Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The presence of additional positively charged patches in the E200K variant could further promote these interactions, placing the protein in an environment that is especially conducive to the transition into the pathogenic PrPSc conformation. Regardless of the specific mechanism, the present data strongly suggest that mutation-dependent surface effects that may lead to abnormalities in cellular interactions of prion protein should be considered key determinants of a spontaneous PrPC → PrPSc conversion in the E200K form of familial CJD. We thank Drs. Chunhua Yuan and Chuck Cottrell for assistance with 800-MHz spectra acquisition and Frank Delaglio, Dan Garrett, and J. P. Linge for helpful comments." @default.
• W2149247478 created "2016-06-24" @default.
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• W2149247478 date "2000-10-01" @default.
• W2149247478 modified "2023-10-11" @default.
• W2149247478 title "Solution Structure of the E200K Variant of Human Prion Protein" @default.
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