Cellular prion protein conformation and functionFred F. Dambergera,1, Barbara Christena,1, Daniel R. Péreza, Simone Hornemanna,2, and Kurt Wüthricha,b,3

aInstitute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zurich, Schafmattstrasse 20, CH-8093 Zurich, Switzerland; andbDepartment of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla,CA 92037

Contributed by Kurt Wüthrich, April 23, 2011 (sent for review March 6, 2011)

In the otherwise highly conserved NMR structures of cellular prionproteins (PrPC) from different mammals, species variations in asurface epitope that includes a loop linking a β-strand, β2, witha helix, α2, are associated with NMR manifestations of a dynamicequilibrium between locally different conformations. Here, it isshown that this local dynamic conformational polymorphism inmouse PrPC is eliminated through exchange of Tyr169 by Ala or Gly,but is preserved after exchange of Tyr 169 with Phe. NMR structuredeterminations of designed variants of mouse PrP(121–231) at20 °C and of wild-type mPrP(121–231) at 37 °C together withanalysis of exchange effects on NMR signals then resulted in theidentification of the two limiting structures involved in this localconformational exchange in wild-type mouse PrPC, and showedthat the two exchanging structures present characteristically differ-ent solvent-exposed epitopes near the β2–α2 loop. The structuraldata presented in this paper provided a platform for currentlyongoing, rationally designed experiments with transgenic labora-tory animals for renewed attempts to unravel the so far elusivephysiological function of the cellular prion protein.

prion disease ∣ protein structure ∣ protein dynamics ∣ transmissiblespongiform encephalopathy

The prion protein (PrP) in its cellular isoform (PrPC), which isfound in healthy mammalian organisms, is among the most

extensively studied proteins. Nonetheless, the physiological func-tion of PrPC and its role in the molecular pathways leading todegeneration of the brain in patients suffering from transmissiblespongiform encephalopathies still remain elusive (for example,refs. 1–3).

The NMR solution structure of the recombinant mouse prionprotein (4, 5) has the same molecular architecture as the proteinpart of PrPC present in healthy organisms (6). It contains a flex-ibly disordered N-terminal tail of residues 23–124, a globulardomain of residues 125–228 with three α-helices and a short two-stranded antiparallel β-sheet, and a short C-terminal tail of resi-dues 229–231 (5) [see Schätzl et al. (7) for the numeration of PrPsfrom different species]. The globular domain in the NMR struc-tures of recombinant PrPs from different mammalian species ishighly similar, except for local structure variability in a surfaceepitope formed in part by a loop of residues 166–172 that con-nects the strand β2 with the helix α2 (4, 8–16). This polypeptidesegment and its immediate spatial environment also show numer-ous amino acid exchanges among mammals (17–19), which con-trasts with the overall high sequence conservation in the globulardomain of mammalian PrPs (7). The β2–α2 loop is structurallydisordered in the NMR structures of numerous mammalian prionproteins determined at 20 °C (4, 9–11), but is well defined inthe NMR structures of PrPC from elk, bank vole, horse, wallaby,and rabbit determined under similar conditions (12–16). Thisstructural disorder is associated with the absence of resonancesfrom residues 167–171 from the β2–α2 loop and residue 175 inthe 2D ½15N;1H�-HSQC spectra suggesting the presence of a localconformational exchange process.

The present paper elucidates a structural basis for rationalizingthe behavior of the surface epitope formed by the β2–α2 loop andthe helix α3 in wild-type mouse PrPC, based on NMR structuredeterminations of two newly designed variants of mPrP(121–231)

at 20 °C and of wild-type mPrP(121–231) at 37 °C. It is thus shownthat the β2–α2 loop structure in mPrPC manifests exchange be-tween just two locally different conformations, and both limitingstructures involved in this conformational exchange are identifiedby combining structural data on wild-type and variant mPrP(121–231) [Protein Data Bank, www.pdb.org (PDB ID codes2L39, 2L40 and 2L1D)] with analysis of exchange effects on loopresidue NMR signals. The insight gained on the solution confor-mations available to mouse PrPC indicate future avenues fordefining the physiological functions of the prion protein.

Results and DiscussionNMR Spectra of mPrP[Y169G](121–231) and mPrP[Y169A](121–231)Show No Evidence of Exchange Line Broadening. In search of a struc-tural basis for possible local conformational exchange on thechemical-shift timescale involving the β2–α2 loop, we designedvariants of mPrP(121–231) containing single amino acid ex-changes in this region. We thus found that 2D ½15N;1H�-HSQCNMR spectra of mPrP[Y169G](121–231) recorded at a 1Hfrequency of 750 MHz in 5° intervals over the temperature range5–40 °C showed no effects of conformational exchange on theNMR line shapes for residues in the β2–α2 loop (Fig. 1A) or re-sidue 175. The same observation was made for mPrP[Y169A](121–231), whereas the line shapes of signals of the loop residuesshow pronounced temperature variation in mPrP[Y169F](121–231) (Fig. 1B), similar to wild-type mPrP(121–231) (Fig. 1C).Hence, either tyrosine or phenylalanine in position 169 maintainsan NMR-observable dynamic local polymorphism in mousePrPC, with conformational exchange at intermediate rates on thechemical-shift frequency timescale, whereas there is no evidencein the NMR data for this conformational polymorphism aftersubstitution of the aromatic residue in position 169 by glycineor alanine. Based on these observations, we determined NMRstructures for the two variants of mPrP(121–231) containingreplacements of Y169 with Gly or Ala, respectively, as well asthe NMR structure of mPrP(121–231) at 37 °C.

NMR Structures of mPrP[Y169G](121–231) and mPrP[Y169A](121–231).For both proteins, nearly complete backbone and side-chainNMR assignments were obtained (for details, see deposits 17081and 17087 at www.bmrb.wisc.edu), using standard techniquesfor uniformly 13C,15N-labeled proteins (20). Statistics of the

Author contributions: F.F.D., B.C., D.R.P., S.H., and K.W. designed research; F.F.D., B.C.,D.R.P., and S.H. performed research; F.F.D., B.C., and K.W. analyzed data; and F.F.D.,B.C., and K.W. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The atom coordinates of the NMR structures of mPrP(121–231) at 37 °Cand of mPrP[Y169A](121–231) and mPrP[Y169G](121–231) at 20 °C have been deposited inthe Protein Data Bank, www.pdb.org (PDB ID codes 2L39, 2L40 and 2L1D), and theirchemical-shift lists have been deposited in the BioMagResBank, www.bmrb.wisc.edu(accession nos. 17174, 17213, and 17081).1F.F.D. and B.C. contributed equally to this work.2Present address: Institute of Neuropathology, University Hospital Zurich, Schmelzberg-strasse 12, 8091 Zurich, Switzerland.

3To whom correspondence should be addressed. E-mail: wuthrich@mol.biol.ethz.ch.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106325108/-/DCSupplemental.

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structure determinations, which used the softwares ATNOS/CANDID (21, 22), DYANA (23), and OPALp (24, 25), are givenin Table 1. The results are visualized in Fig. 2 A and B and Fig. S1,with the amino acid side chains in Fig. S1 color-coded accordingto their global displacement values, D (26). The architecture ofthe two proteins coincides with the structures of other mamma-lian PrPs (4, 8–16), consisting of three α-helices spanning residues144–153, 172–190, and 200–226, and a short antiparallel β-sheetof residues 128–131 and 161–164, where the helices α1 and α2terminate with 310-helical turns of three to four residues. Withinthis conserved structural scaffold, the β2–α2 loop (located belowthe number 172 in the two structures shown in Fig. 2 A and B)adopts a different structure from that seen in all other PrPCs stu-died so far, with the residues 167–170 forming a type I β-turn(Fig. 3) (27, 28).

NMR Structure of Wild-Type mPrP(121–231) at 37 °C. An NMR struc-ture determination of wild-type mPrP(121–231) was pursued un-der conditions where the amide proton NMR signals of all β2–α2loop residues are observable (see Fig. 1C). Tests of the stabilityof a 1.5 mM NMR sample of ½u-15N�-mPrP (121–231) in 10 mMsodium acetate buffer, pH 4.5, at variable temperature by mon-itoring the signal intensities in 1D 1H NMR spectra showed thatat 37 °C there was a loss due to deterioration of the protein solu-tion of only about 5% after 4 d. We therefore recorded each ofthree 3D heteronuclear-resolved ½1H;1H�-NOESY spectra with afreshly prepared 1.5 mM solution of uniformly 13C,15N-labeledmPrP(121–231) at 37 °C. A 3D 15N-resolved ½1H;1H�-NOESYdataset recorded at 500 MHz, where the lower 1H resonance fre-quency was chosen to obtain narrower line widths, yielded NOEcross-peaks with the amide protons of all the β2–α2 loop residues.Based on nearly complete resonance assignments (deposit 17174at www.bmrb.wisc.edu), the NMR structure was determined(Table 1).

The globular fold of mPrP(121–231) at 37 °C (Fig. 2C) isclosely similar to that of mPrP(121–231) at 20 °C (4, 12) and thestructures in Fig. 2 A and B, with an antiparallel two-strandedβ-sheet of residues 128–131 and 161–164, three α-helices com-

prising the residues 144–153, 172–190, and 200–227, and 310-helical turns formed by the residues following the helices α1 andα2 (Fig. 2C). The β2–α2 loop structure includes a well-defined310-helical turn for residues 166–169, which is different from boththe loop structure in the variant proteins of Fig. 2 A and B, andthe structurally disordered loop in the mPrP(121–231) structureat 20 °C (4, 12).

Survey of β2–α2 Loop Conformations in NMR Structures of Mamma-lian PrPCs. In this section, we compare the conformations of thepolypeptide segment 165–175 in NMR structures of recombinantmammalian PrPCs and designed variants of mPrPC (Fig. 3 andFig. S2). Using the standard criteria implemented in the programMOLMOL (29), we searched these structures for regular second-ary structure elements and the presence of hydrogen bonds, andwe evaluated the deviations of the 13Cα chemical shifts from therandom coil values, Δ13Cα, for the individual residues.

In all NOE-based NMR structures of PrPCs that containTyr in position 169 and show a well-defined β2–α2 loop, the re-sidues 166–169 form a 310-helical turn, with hydrogen bondsHN∕Hε168–O165 and HN169–O166 (Fig. 3 B–F). The 310-helicalturn is also manifested by a typical pattern of Δ13Cα values, with alarge positive Δ13Cα value for Val166 and smaller positive Δ13Cα

values for the residues 167 and 168. This loop conformation isobserved in the wild-type mPrP(121–231) structure at 37 °C(Fig. 3B) and in all PrPCs that show a well-structured β2–α2 loop

A

B

C

Fig. 1. Temperature dependence over the range 5–40 °C of the NMR signalsof the β2–α2 loop residues 167–171 in two designed variants of mPrP(121–231) generated by exchange of residue Y169 and in wild-type mPrP(121–231). Cross-sections along ω2ð1HÞ from 750 MHz 2D ½15N;1H�-HSQCspectra are shown. (A) mPrP[Y169G](121–231). (B) mPrP[Y169F](121–231).(C) mPrP(121–231). The signal of A133 represents the behavior of those re-sidues that are not affected by intramolecular conformational exchange.

Table 1. Input for the structure calculation and characterization ofthe energy-minimized NMR structures of mPrP[Y169G](121–231)and mPrP[Y169A](121–231) at 20 °C and mPrP(121–231) at 37 °C(“WT at 37 °C”)

Y169G Y169A WT at 37 °C

ConstraintsNOE distance limits* 3,107 2,901 3,298

Intraresidual 616 644 608Sequential 832 762 846Medium-range 893 817 938Long-range 766 678 908

Dihedral angles 128 126 116Target function, Å2 1.60 ± 0.39 2.31 ± 0.20 2.43 ± 0.59

Residual violationsNOEs

Number >0.1 Å 34 ± 5 31 ± 5 32 ± 6Maximum (Å) 0.15 ± 0.01 0.14 ± 0.02 0.14 ± 0.01

Dihedral anglesNumber >2.0° 0 ± 0 0 ± 0 0 ± 0Maximum, ° 1.17 ± 0.48 1.54 ± 0.61 1.56 ± 1.83

Amber energies, kcal · mol−1

Total −4,892 ± 49 −4,922 ± 105 −4,975 ± 92van der Waals −350 ± 11 −322 ± 12 −338 ± 16Electrostatic −5,469 ± 42 −5,554 ± 94 −5,577 ± 82

Rmsd, ņ

bb (125–226) 0.39 ± 0.06 0.49 ± 0.06 0.39 ± 0.05ha (125–226) 0.72 ± 0.05 0.89 ± 0.05 0.70 ± 0.05

Ramachandran statistics, %‡

Most favored 80.5 81.4 80.8Additional allowed 16.7 16.6 16.9Generously allowed 1.5 1.9 2.2Disallowed 1.4 0.3 0.1

Except for the entries describing the input for the structure calculations,the average values for the 20 energy-minimized conformers with thelowest residual DYANA target function values and the standard deviationsamong them are given.*The different numbers of experimental upper distance limits are primarilydue to somewhat different protein concentrations used to record the NMRdata for the individual PrPs.

†The rmsds were calculated relative to the mean coordinates for the bundleof 20 conformers. bb indicates the backbone atoms N, Cα, C′; ha stands for“all heavy atoms.” The numbers in parentheses indicate the residues forwhich the rmsd values were calculated.

‡As determined by PROCHECK (40).

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at 20 °C, including the wild-type proteins of elk, bank vole,wallaby, and horse (Fig. 3 C–F and Fig. S2) (12–15). Because the13Cα NMR signals of all β2–α2 loop residues except Asp167 areobservable in mPrP(121–231) also at 20 °C, and the measuredΔ13Cα values are the weighted average over the Δ13Cα valuesof all the conformers involved in the exchange process that leadsto line broadening of amide group signals (Fig. 1 B and C), thepattern of Δ13Cα values in Fig. 3A implies that a 310-helical turn isthe dominantly populated β2–α2 loop conformation also in mPrP(121–231) at 20 °C.

The type I β-turn conformation of the β2–α2 loop observedin the NMR structures of mPrP[Y169A](121–231) and mPrP[Y169G](121–231) (Fig. 2 A and B), with hydrogen bondsHN167–Oγ170 and HN170–O167 (Fig. 3 G and H), has so faronly been observed in mPrPC variants with glycine or alanine inposition 169. The Δ13Cα values show a different pattern from thatobserved for the 310-helical turn, with residue 168 having a largepositive Δ13Cα value, residue 166 a somewhat smaller positivevalue, and residue 167 a value near zero.

Conformational Exchange Between 310-Helical and Type I β-Turn Con-formations of the β2–α2 Loop Causes NMR Line Broadening in Wild-Type Mouse PrPC. The line narrowing observed in loop signals of

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Fig. 2. NMR structures at 20 °C and pH 4.5 of designed variants of mPrP(121–231) generated by exchange of Y169 and of mPrP(121–231) at 37 °C. (A) mPrP[Y169G](121–231). (B) mPrP[Y169A](121–231). (C) mPrP(121–231) at 37 °C.Shown are bundles of 20 energy-refined DYANA conformers representingthe polypeptide backbone of residues 125–228 by a spline function throughthe Cα atoms. The sequence locations at the start and end of the threeα-helices are indicated. Color code: β-strands, green; 310-helical turns, cyan;α-helices, red (A), orange (B), and yellow (C). All-heavy-atom stereo views ofstructures from Fig. 2 A and B are displayed in Fig. S1.

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165 170 175

Fig. 3. NMR structures and 13Cα chemical shifts of the polypeptide segment165–175, which includes the β2–α2 loop, in eight PrP(121–231) constructs. Alldata were collected at pH 4.5. The temperature was 20 °C, except for B, D,and F. (A) Wild-type mPrP. (B) Wild-type mPrP at 37 °C. (C) Elk PrP. (D) HorsePrP at 25 °C. (E) mPrP[S170N]. (F) mPrP[D167S] at 25 °C. (G) mPrP[Y169G]. (H)mPrP[Y169A]. Shown are the polypeptide backbone as a spline functionthrough the Cα atoms, with the radius of the tubes proportional to the meanglobal backbone displacement per residue among a bundle of 20 energy-minimized conformers used to represent the NMR structures, the hydrogenbonds as dashed yellow lines, and the 13Cα chemical-shift deviations from therandom coil values, Δ13Cα, where red color highlights data associated withthe 310-helical conformation observed in B–F, and blue color those for theβ-hairpin observed in G and H. The hydrogen bonds are HN168–O165 andHN169–O166 in B–F, and HN170–O167 and HN167–Oγ170 in G and H. In A,the 13Cα chemical-shift data are incomplete because of a missing assignmentfor residue 167, and because the loop is structurally disordered, no hydrogenbonds are indicated (see text). The atom coordinates were taken from thefollowing PDB deposits: (A) PDB ID code 2L1H, (B) PDB ID code 2L39, (C)PDB ID code 1XYW, (D) PDB ID code 2KU4, (E) PDB ID code 2K50, (F) PDBID code 2KU5, (G) PDB ID code 2L1D, and (H) PDB ID code 2L40.

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mPrP(121–231) upon raising the temperature suggest that eachamide resonance is the average of multiple conformers that inter-convert rapidly relative to their chemical-shift difference. Theline shapes of NMR signals averaged by conformational exchangedepend on the exchange rate constant, k; the populations of theexchanging states; and the difference between the chemical shifts(in frequency units) in the exchanging structures, Δν. The resultsdescribed in the following reveal that the NMR data for wild-typemouse PrPC can be explained by exchange between two structurallycharacterized conformers, A and B, with populations pA and pB,and k ≫ Δν. The contribution from the conformational exchangeto the observed line widths at half height, Δν1∕2, then is

Δν1∕2 ¼ 2 · π · pA · pB · ðΔνÞ2 · k−1; [1]

where Δν1∕2 and Δν are given in Hz, and k is in s−1 (30, 31). For acommon set of k, pA and pB values, the NMR signals of the indi-vidual exchanging residues may be differently broadened, due todifferent Δν values (32). Consistent with the prediction of Eq. 1that the line broadening is enhanced at increasing spectrometerfield because of increasing Δν, broader lines were observed forthe loop residues at 750 MHz than at 500 MHz (Fig. S3A). Forthis reason, we measured the 15N-resolved ½1H;1H�-NOESY usedfor the structure determination of mPrP(121–231) at 500 MHz(Fig. S3B).

Based on Eq. 1, the experimental evidence supports that theNMR line broadening of β2–α2 loop residues in wild-type mPrPC

is due to exchange between two conformations that contain, re-spectively, the 310-helix or the type I β-turn loop structure (Fig. 3).Thus, the residues 170, 171, and 175, which show the largestamide proton chemical-shift differences, Δνð1HÞ, between thetwo limiting conformations, which are represented here by thechemical shifts at 40 °C of wild-type mPrP(121–231) and the de-signed variant mPrP[Y169A](121–231), respectively, also showthe largest line-broadening effects along ω2ð1HÞ, Δν1∕2ð1HÞ, inmPrP(121–231) (Fig. 4 A and B), whereas there is no exchangeline broadening for these residues in mPrP[Y169A](121–231)(Fig. 4C). A similar qualitative correlation is seen for residues167 and 171, which show large 15N chemical-shift differences,Δνð15NÞ, and large line broadening along ω1ð15NÞ) in mPrP(121–231) (Fig. S4) where as no line broadening is observed inmPrP[Y169A](121–231). Furthermore, there is no loop residuewith small Δν values along ω1ð15NÞ or ω2ð1HÞ that shows visibleexchange broadening of its NMR signals. These qualitativecorrelations between corresponding Δν and Δν1∕2 values havebeen quantitatively substantiated through verification of thelinear relation between Δν1∕2 and Δν2 predicted by Eq. 1 as de-scribed in Materials and Methods (Fig. 4D). The data points forresidues 166, 168, and 172–174 (Fig. 4 A–C and Fig. S3) wouldalso fall on the linear regression curve of Fig. 4D, near its origin atΔν2 ¼ 0 and Δν1∕2 ¼ 0.

Estimates for the values of the parameters pA, pB and k in Eq. 1resulted from the following considerations: The slope of the lin-ear regression curve in Fig. 4D corresponds to a value of 9;000 s−1for the product k∕ð2π · pA · pBÞ in Eq. 1. Using some simplifyingassumptions for the experimental determination of Δν and con-sidering that the largest possible value of the product pA · pB is0.25, k ≤ 14;000 s−1 is obtained as an estimate for the upper limitof the exchange rate constant. A more conservative treatment in-dicates a limiting value of k ≤ 56;000 s−1 (see SI Text). A lowerlimit of k ¼ 800 s−1 has been derived from the fact that the lar-gest Δν values affecting the exchange-averaged signals of the twoconformations were about 800 Hz at the 1H resonance frequencyof 750 MHz used for these measurements (Fig. 4A). The corre-sponding lower limit for the population of the minor species ispB ¼ 0.014.

In conclusion, the data presented in the preceding sectionslead to the following description of the conformational state

of wild-type mouse PrP(121–231) in solution: mPrPC forms twolocally different and widely unequally populated structures. Themore abundant species contains a 310-helix structure of the β2–α2loop and its population is above 90%, and the lesser populatedspecies includes a type I β-turn structure of the β2–α2 loop. Overthe temperature range of 20–40 °C the two structures exchange atintermediate rates on the chemical-shift timescale, as manifestedin the signal line shapes of the NMR spectrum (Fig. 1C). Themajor structure involved in this exchange process on the millise-cond to microsecond timescale represents an ensemble of rapidlyexchanging conformers that all contain the 310-helix loop struc-

1

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[Hz2]X105

Fig. 4. Proton chemical-shift differences between corresponding 750 MHz2D ½15N;1H�-correlation NMR signals in mPrP(121–231) and mPrP[Y169A](121–231) at 40 °C, and exchange broadening of the NMR signals. (A) Histo-gram-type plot versus the mPrP(121–231) amino acid sequence of the squareof the amide proton shift differences between the two proteins in hertz,Δνð1HNÞ2. (B) Plot of the linewidths at half-height in mPrP(121–231) alongω2ð1HÞ,Δν1∕2ð1HNÞ. (C) Same as B for mPrP[Y169A](121–231). (D) Quantitativeassessment, using Eq. 1, of the hypothesis that the NMR line broadening forβ2–α2 loop residues in mPrP(121–231) is due to exchange between the 310-helix and β-hairpin conformations of the β2–α2 loop. 1H and 15N NMR linebroadening is plotted along the horizontal axis, and the square of chemi-cal-shift differences between corresponding NMR signals in mPrP(121–231)and mPrP[Y169A](121–231) is plotted along the vertical axis. Data are shownonly for those signals of residues 166–175 that show large line broadening,with 1HN data (Fig. 4 A and B) indicated by squares and 15N data (Fig. S4 Aand B) by crosses. The horizontal axis is in units of hertz and the vertical axisin squared hertz. The slope of the plot representing k∕ð2π · pA · pBÞ in Eq. 1 isequal to 9;000 s−1. The Pierson correlation coefficient is 0.95. The chemicalshifts used here were obtained by transferring the resonance positions fromthe available assignments for mPrP(121–2321) at 37 °C (BMRB accession no.17174) and mPrP[Y169A](121–231) at 20 °C (BMRB accession no. 17213) tothose at 40 °C by experiments of the type shown in Fig. 1.

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ture (Fig. 3B), and the less populated structure represents asimilar ensemble of conformers that all contain the type I β-turnstructure of the loop (Fig. 3H).

Biological Implications. A possible link between the data ofFigs. 1–4 and physiological functions of the cellular prion proteinresulted from a search of the database of all available prion pro-tein amino acid sequences (see Materials and Methods), whichshowed that Tyr169 is strictly conserved in mammalian species.Although it maintains both the local exchange process in theβ2–α2 loop of mPrP[Y169F](121–231) (Fig. 1B) and the overallstability of the PrPC fold, Phe169 is not encountered in mamma-lian PrPs. There is thus an indication that the hydroxyl group oftyrosine in position 169 has an essential role for the physiologicalfunction of mammalian prion proteins.

Comparison of mPrP(121–231) (Fig. 5A) and mPrP[Y169A](121–231) (Fig. 5B) shows that different amino acid side chainsare exposed to the solvent in the two structures. Firstly, althoughthe hydroxyl function of Ser170 is part of the surface epitope inthe 310-helix loop structure, it is buried in the protein interior inthe type I β-turn conformation. Secondly, the side chain of resi-due 169 changes from a location in the 310-helical loop structure,where it points toward the protein core, to a surface-exposed or-ientation in the β-turn conformation of the β2–α2 loop. Modelingstudies based on replacing Ala169 in the structure of Fig. 5B byTyr indicate that if the β2–α2 loop in mammalian mPrPCs takes onthe type I β-turn conformation, the side chain of Tyr169 is ex-posed on the protein surface (Fig. 5 C and D). From combinedanalysis of the exchange broadening of the NMR signals (Figs. 1Band 4 A–D) and the 13Cα chemical shifts, and in particular fromthe fact that the patterns of 13Cα chemical shifts in the two formsof the β2–α2 loop are characteristically different (Fig. 3 andFig. S2), we conclude that in wild-type mPrPC the 310-helix con-formation of the loop is the more abundant form, with estimatedpopulations of ≥0.9 and ≤0.1 for the two locally different con-formations (see also the preceding section and SI Text).

It will now be of keen interest to follow up on these structuralstudies with experiments using transgenic mice expressing variantmouse prion proteins that are devoid of the tyrosine hydroxylgroup in position 169. In this context, one should also recall thatearlier work advanced the suggestion, which has so far not beensubstantiated, that a surface epitope formed in part by the β2–α2loop in PrPC might be a recognition site for effector moleculesthat would affect the transition from the cellular form of the prionprotein to the disease-related, aggregated PrPSc form (33, 34).

Materials and MethodsProtein Preparation. Clones for mPrP[Y169A](121–231), mPrP[Y169F](121–231), and mPrP[Y169G](121–231) were obtained by single-amino acid substi-tutions in the mPrP(121–231) gene, using the QuikChange® site-directedmutagenesis kit (Stratagene). Uniformly 15N- and 13C,15N-labeled proteinswere prepared as described (9, 35, 36). For the NMR experiments, concen-trated solutions containing 1 to 2 mM protein were prepared in H2O contain-ing 10 mM [d4]-sodium acetate buffer at pH 4.5, 10% D2O, 0.02% sodiumazide and a protease inhibitor cocktail (Roche).

NMR Experiments. NMR data for the structure determinations of mPrP[Y169A](121–231) and mPrP[Y169G](121–231) were measured at 20 °C, thosefor mPrP(121–231) at 37 °C. The presence of the PrPC fold in mPrP[Y169F](121–231) was verified by the close similarity of the 2D ½15N;1H�-HSQC spec-trum to that of mPrP(121–231). Resonance assignments were obtained withstandard triple resonance experiments (20) recorded on a Bruker DRX500spectrometer with cryogenic probehead. Three-dimensional 15N-resolved½1H;1H�-NOESY and 13C-resolved ½1H;1H�-NOESY spectra were recorded onBruker Avance900 and DRX750 spectrometers, using a mixing time of 60 ms,except that the 3D 15N-resolved ½1H;1H�-NOESY data for mPrP(121–231) at37 °C was recorded at 500 MHz in order to reduce the exchange line broad-ening. Chemical shifts for mPrP(121–231) were referenced relative to internal2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), and for the other two pro-teins with a coaxial insert (Norell Inc.) containing DSS in NMR buffer, because

of protein precipitation upon addition of DSS. The program CARA (37)(www.nmr.ch) was used for the analysis of the NMR spectra.

The temperature-dependence of 2D ½15N;1H�-HSQC spectra was measuredat 750 MHz with 1 mM solutions of the 15N-labeled proteins. Prior to Fouriertransformation, the datasets were zero-filled to 16 k and 1 k points in the 1Hand 15N dimensions, respectively, and linewidths were measured at half theheight of the maximum signal intensity. The exchange contribution to theobserved linewidths was estimated by subtracting the average linewidthdetermined from all signals excluding those deviating by one standard de-viation from the mean value. These experiments were also used to measurethe chemical shifts of mPrP(121–231) and mPrP[Y169A](121–231) at 40 °C,which have been used in Fig. 4 and Fig. S4.

A

B

C

D

Fig. 5. Protein surface epitopes formed in the NMR structures by residues164–175 in mPrP(121–231) at 37 °C and mPrP[Y169A](121–231) at 20 °C,which represent the two limiting structures connected by intermediate-rate conformational exchange in wild-type mammalian PrPCs (see text).The polypeptide backbone from the end of strand β2 at residue 163 to thesecond turn of helix α2 at residue 178 is shown, with space-filling presenta-tions of the side chains 164 and 171 in blue, 168–170 in functional colors,and 175 in green. (A) NMR structure of mPrP(121–231) at 37 °C. (B) NMRstructure of mPrP[Y169A](121–231) at 20 °C. (C and D) Models of the less-abundant conformation of mPrP(121–231) (see text) generated by replace-ment of A169 in the experimental structure (B) by Y169; only two of thethree staggered rotamers about the Cα–Cβ bond are shown, withX1 ¼ þ60° (C) and X1 ¼ −60° (D), because the third rotamer would involveextensive steric crowding.

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NMR Structure Calculation. The standard protocol of the stand-alone ATNOS/CANDID program package (21, 22), version 1.2, was used together withDYANA (23) for automatic peak picking, automatic NOE assignment, andstructure calculation. The final cycle of the calculation was started withbetween 80 and 120 randomized conformers, and the 20 conformers withthe lowest residual target function values were energy-minimized in a watershell with the program OPALp (24, 25), using the AMBER force field (38).The program MOLMOL (29) was used to analyze the results of the proteinstructure calculations, including regular secondary structure identificationwith the method of Kabsch and Sander (39).

Database Search of Residue Types in the Sequence Position 169 of MammalianPrion Proteins. Mammalian PrP sequences deposited as of December 2010

were analyzed to determine residue variations in position 169, using the Uni-ProtKB (Protein Knowledgebase; www.uniprot.org) with the sequence ofmPrP(1–254) as input. Nonmammalian PrP sequences as well as mammalianPrP sequence fragments that do not contain information on position 169[according to the numeration used by Schätzl et al. (7)] were removed fromthe list, which finally contained 524 PrP sequences from 213 different mam-malian species.

ACKNOWLEDGMENTS. This work was supported by the Swiss National ScienceFoundation and the Eidgenössische Technische Hochschule Zurich throughthe National Center of Competence in Research “Structural Biology,” andby the European Union (Understanding Protein Misfolding and Aggregationby NMR, project no. 512052).

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