Structural details of amyloid beta oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy

Human PrP (huPrP) is a high-affinity receptor for oligomeric Aβ. Synthetic oligomeric Aβ species are known to be heterogeneous, dynamic and transient, rendering their structural investigation particularly challenging. Here, we used huPrP to preserve Aβ oligomers by co-precipitating them into large hetero-assemblies to investigate the conformation of Aβ(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the last two α-helices. For Aβ(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal a substantial β-strand content. Importantly, not all Aβ(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aβ fibrils suggests that the Aβ oligomer preparation represents a heterogeneous mixture of β-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aβ to adopt variable β-structure conformers.


Introduction
Alzheimer's disease (AD) accounts for an estimated 60 to 80 % of all types of dementia 1 .
One of the hallmarks of AD is the formation of amyloid plaques, which consist mainly of amyloid β (Aβ) peptides comprising 39 to 43 residues 2 . Aβ is produced by cleavage of the amyloid precursor protein (APP) by β-and γ-secretases 3 . Of the two most abundant species Aβ(1-40) and Aβ(1-42), the latter is more prone to aggregation and its aggregates are more toxic 3 . Small to moderately sized Aβ oligomers (Aβoligos) have been identified as the most neurotoxic factor in the pathogenesis of AD, whereas large fibrils are known to be the main component of insoluble plaques 4 . Detailed structural information on Aβ(1-42)oligo is still missing due to their transient nature and probably also due to high structural variability between different or within the same oligomer preparations. Heterogeneity and dynamic behavior concerning sizes and conformations is a major challenge to structural studies of oligomers 5 . This challenge has previously been met by freeze-trapping 6,7 , by applying special aggregation conditions 8 , by the addition of stabilizing compounds 9 or antibodies 10 , or by engineered mutagenesis 11 . Here, we used the recombinant human prion protein, in its native PrP C conformation to trap Aβ oligomers by co-precipitating them into large heteroassemblies, in which the growth of Aβ oligomers is prevented, as demonstrated by long-term solid-state NMR measurements over 11 months.
PrP C is a high-affinity cell-surface receptor for Aoligo 12,13 as well as for fibrillar A [14][15][16] . It has been suggested that binding of Aβoligo to membrane-anchored PrP C mediates Aβ toxicity during AD by mediating synapse damage 17 and the blockade of long-term potentiation by Aβoligo 12,18 via activation of Fyn-kinase pathways 19,20 , but this has also been questioned [21][22][23][24] . It has also been described that soluble PrP 25 as well as its N-terminal fragment PrP   26,27 have a protective role by inhibiting Aβ fibrillation and formation of Aβoligo.
Several in vitro studies on the Aβ-PrP interaction suggest that Aβoligos bind at two Lys-rich parts (residues 23 to 27 and ≈ 95 to 110) on PrP [28][29][30][31][32][33] , but an additional involvement of the Cterminus of PrP has also been suggested 14 . A structural study of insoluble PrP C -Aβoligo complexes described this as a "hydrogel", in which the A oligomers were rigid, while PrP still has high molecular mobility 34 . Additionally, this study reported a conformational change in the N-terminus of PrP C upon complexation with Aβoligo. We recently demonstrated that Aβoligo forms large hetero-assemblies with either full-length (huPrP(23-230)) or C-terminally truncated (huPrP(23-144)) membrane-anchorless monomeric PrP 33 . These assemblies have a size of a few micrometers as determined by dynamic light scattering and show cloud-like morphologies as seen by atomic force microscopy 33 . The Aβ:huPrP stoichiometry of the hetero-assemblies depends on the PrP concentration added to Aβoligo and reaches a value of 4:1 (monomer ratio) in the presence of an excess of either huPrP  or huPrP   33 .
In all these in vitro preparations A oligomers and early stage protofibrils are stabilized and prevented from elongation by PrP, which has been shown to preferentially bind to fast growing fibril and oligomer ends 15 .
Here we exploit this stabilizing effect in an NMR study on different samples of Aβoligo complexed by huPrP. Isotope labeling of either huPrP or A allowed us to characterize both components of the complex separately. While the N-terminal region of huPrP in the complex remains largely devoid of secondary structure and still undergoes fast backbone conformational averaging on the s to ms time scale, Aoligos exhibit a high degree of strand conformation. While these Aoligos are highly heterogeneous, solid-state NMR spectra reveal similarities with corresponding spectra of all fibril polymorphs published so far [35][36][37] .

The N-terminal construct huPrP(23-144) is disordered in solution at mildly acidic and neutral pH
The solution structure of huPrP(23-230) had originally been determined in acetate buffer at an acidic pH of 4.5 and 20 °C 38 , whereas the huPrP-Aβ(1-42)oligo complex samples for solidstate NMR were prepared at a pH value close to neutral. As a basis for studying the interaction between huPrP and Aβoligo we therefore first investigated free huPrP(23-144) by NMR spectroscopy in solution at different pH values ranging from 4.5 to 7.0 and at a temperature of 5.0 °C, which is closer to the temperature used for the solid-state NMR experiments. As reported previously, the chemical shifts of the N-terminal amino acid residues 23 to 124 in truncated huPrP  are almost identical to those of huPrP , whereas residues 125 to 144, which are part of the well-ordered globular domain of huPrP , are strongly affected by the truncation at position 144 33 .
We obtained almost complete sequence-specific 1 H, 13 C, and 15 N backbone resonance assignments for huPrP  at pH values of 4.5 and 7.0 and a temperature of 5.0 °C using a combination of HNCO, HNCACB, and BEST-TROSY-(H)N(COCA)NH triple-resonance experiments (Supplementary Fig. 1). The assigned chemical shifts at pH 4.5 and pH 7.0 have been deposited with the Biological Magnetic Resonance Data Bank (BMRB) under accession codes 28115 and 28116, respectively.
As expected, side-chain titration in this pH range causes significant chemical shift changes for all 7 histidine residues and for residues next to histidine. Other than that, the chemical shifts at pH 4.5 and pH 7.0 are very similar to each other and very close to random coil shifts 39 . Quantitative analysis reveals that the Random Coil Index (RCI) order parameters 40 SRCI 2 , which are a measure of how different the backbone chemical shifts are from those of a disordered random coil on a scale of 0 (typical for a random coil) to 1 (typical for a wellordered backbone conformation), are consistently below ≈ 0.6 ( Supplementary Fig. 2). This demonstrates conclusively that free huPrP  in solution at neutral and mildly acidic pH is highly disordered and devoid of any stable secondary structure.

The flexible N-terminus of huPrP becomes immobilized but remains almost devoid of regular secondary structure upon binding to Aβoligo
Formation of high molecular weight assemblies was analyzed by sucrose density gradient ultracentrifugation (DGC) and subsequent SDS-PAGE, and RP-HPLC 33 ( Supplementary   Fig. 3). This indicates that the potential PrP binding sites on Aβoligo are not yet saturated with huPrP(23-144). As previously described 33 , a molar ratio of Aβ:PrP of 4:1 is obtained in the precipitate if huPrP is added in excess.
To probe the flexibility of the N-terminal construct huPrP  in the complex, we recorded a 1 H-13 C Insensitive nuclei enhanced by polarization transfer (INEPT)-NMR spectrum as well as dipolar based 1 H-13 C and 1 H-15 N CP-MAS spectra 41 . The INEPT-NMR spectrum of this sample did not show any protein signals at a temperature of ≈ 27 °C (spectrum not shown), whereas in 1 H-13 C and 1 H-15 N CP spectra obtained at ≈ 0 °C strong signals typical for all amino acid types can be seen (Supplementary Fig. 4). This indicates that huPrP  in complex with Aβ(1-42)oligo is immobilized and does not undergo rapid isotropic reorientation as in solution.
In Fig. 1 a typical 2D 13 C-13 C-correlation spectrum obtained with proton driven spin diffusion (PDSD) of huPrP(23-144)*-Aβ (* indicates that the huPrP moiety of the complex is 13 C/ 15 N labelled) is overlaid with a 13 C-13 C total correlation spectrum (TOCSY) of monomeric huPrP  in solution at pH 6.7. Except for some Val and Ala resonances, most of the peaks align well. This indicates that the natively unfolded N-terminus of huPrP does not undergo a major conformational rearrangement upon complex formation with Aβoligo, but conformational averaging of backbone conformations is still possible on the µs to ms time scale. Due to the lack of secondary structure in the intrinsically unstructured N-terminus as well as the repetitiveness of the amino acid sequence in the octarepeats the signal overlap is so severe that sequence-specific resonance assignment for the solid-state NMR spectra was not possible.

Fig. 1
Comparison of a PDSD spectrum of huPrP(23-144)*-Aβ (* species is 13 C, 15 N uniformly labeled) in red with a 13 C-13 C TOCSY spectrum of monomeric huPrP(23-144) in black. The PDSD spectrum was recorded at a temperature of ≈ -6 °C, a spinning frequency of 11 kHz and a mixing time of 30 ms and the TOCSY spectrum at a temperature of 5 °C, at pH 6.7. Grey circles indicate some identified amino acid types, dashed lines Pro and Val connections in the PDSD spectrum. Due to broad line widths and a low signal dispersion in the PDSD spectrum several correlations overlap, especially for the residues in the octarepeat region. Nevertheless, spin systems for most of the amino acid types present in the sequence could be identified and an amino acid-type specific resonance assignment was possible.
Differences between the PDSD and TOCSY spectrum are highlighted with blue circles. For an additional PDSD spectrum see Supplementary Fig. 5, the corresponding double-quantum single quantum correlation spectrum (DQ-SPC5) is shown in Supplementary Fig. 6.
While most of the resonances of huPrP  in complex with Aβoligo align well with those of huPrP  in solution, some differences can be clearly seen; in particular, some Ala, Val and Leu resonances are shifted from random coil towards α-helical secondary chemical shifts. Six out of seven Ala residues as well as both Val and Leu residues present in the sequence are located within a short stretch from residue 113 to 130, a region which starts with the so-called palindrome segment (A 113 GAAAAGA 120 ) (see Fig. 2a). Thus, structural changes upon complexation with Aβoligo in N-terminal huPrP seem to be confined to the region between A113 and L130.  38 . The Aβ-binding regions K23 to K27 and T95 to K110 [28][29][30][31][32][33] and the five octarepeats are indicated above the sequence. b 3D structure of the natively folded prion domain (residues 125 to 228) of full-length huPrP(23-230) in solution. β-strands are colored blue, α-helices red. Picture adapted from PDB-File 1QLZ 38 .
Residues whose entire spin system is missing or shifted in the PDSD spectra ( Fig. 1) are highlighted in purple in a and b.
To exclude self-aggregation of huPrP

The C-terminus of huPrP shows changes in α-helices 2 and 3 upon Aβoligo binding
For full-length huPrP in complex with Aβoligo, huPrP(23-230)*-Aβ (see Table 1) no signals were detected at ≈ 30 °C in the INEPT spectrum (not shown), which indicates that not only the N-terminus, but also the C-terminus of huPrP is not highly dynamic. By contrast, a 1 H-13 C CP spectrum recorded at ≈ 0 °C displays the full signal set expected for a protein ( Supplementary Fig. 8).
Again a line width of ≈ 1 ppm is observed for the 13 C resonances, and due to the large number of resonances and the limited signal dispersion, the signal overlap is so substantial, that a sequential resonance assignment or even a quantitative analysis of residue-specific correlations was not possible. Nevertheless, a comparison with the corresponding 2D 13 C-13 C correlation spectrum of the N-terminal construct huPrP  in complex with oligomeric Aβ (red outline in Fig. 3) allows some conclusions about the structure of full-length complexed huPrP: First, almost all resonances observed in the spectrum of C-terminally truncated huPrP(23-144) appear to be also visible in the spectrum of full-length huPrP(23-230)*-Aβ (Fig. 3). These findings suggest that the C-terminus of full-length huPrP(23-230) does not have a major impact on the conformation of the N-terminus and its interaction with Aβoligo, in line with previous results [28][29][30]33 . Second, the spectrum of fulllength huPrP complexed by Aβoligo displays additional resonances which are absent in the 9 spectrum of N-terminal huPrP  in complex with Aβoligo. Some of the amino acid residues occurring mainly in the C-terminus (e.g. Ile, Thr and Val) give rise to crosscorrelation signals that can be unambiguously identified in 2D 13 C-13 C cross-correlation spectra. However, for most C-terminal amino acid residues (e.g. Asp, Glu, Tyr etc., Fig. 2a) the 2D correlations overlap with other resonances and can therefore not be unambiguously assigned. Further, higher flexibility of the C-terminus of huPrP as compared to the Nterminus may result in reduced signal intensity for amino acid residues from those regions.

Fig. 3
Comparison of two PDSD spectra of huPrP(23-230)*-Aβ (* species is 13 C, 15 N uniformly labeled), shown in black, and huPrP(23-144)*-Aβ, shown as red contour. Both spectra were recorded at a spinning frequency of 11 kHz and a mixing time of 30 ms, but the black one at a temperature of ≈ 0 °C and the red one at ≈ -6 °C.
We compared our 2D 13 C-13 C correlation spectrum with the expected correlations between the chemical shifts obtained experimentally for natively folded full-length huPrP in solution at pH 4.5 38 . While the predicted N-terminal cross peaks (residues 23 to 124) superimpose well with the spectrum of huPrP(23-230)*-Aβ, some discrepancies between the experimental and the predicted spectrum are observed for the C-terminus (residues 125 to 230) ( Supplementary Fig. 9).
In particular, correlation signals for Ile, Thr and Val in α-helical conformation from α-helices 2 and 3 in natively folded huPrP are completely missing in the experimental spectrum ( Supplementary Fig. 10). Instead, correlation signals for Thr and Val with secondary chemical shifts indicative of β-strands that are not observed in natively folded huPrP are clearly visible in the experimental spectrum ( Supplementary Fig. 9 to 11). This suggests that at least for a substantial fraction of the huPrP molecules within the complex some parts of a region between either V121 and I139 and/or V176 and I215 (located in α-helices 2 and 3) have undergone some structural rearrangements including β-strand formation (Fig. 2b).

High β-strand content of Aβoligo in huPrP(23-144)-Aβ complexes
We also investigated the homogeneity and structural characteristics of Aβoligo using two samples containing uniformly 13 C, 15 N labeled Aβoligo in complex with non-labeled huPrP  in different molar ratios (Table 1).
INEPT spectra recorded at ≈ 20 °C of both samples are devoid of protein signals (not shown), whereas 1 H-13 C and 1 H-15 N CP spectra recorded at ≈ 0 °C display strong signals typical for all amino acid residue types (Supplementary Fig. 12). These findings indicate that also the Aβ molecules are rigid parts of the complex. In all 2D and 3D homonuclear 13 C-13 C and heteronuclear 15 N-13 C correlation spectra (see Fig. 4 and Supplementary Fig. 13 to 17) linewidths are rather broad (0.9 ppm for 13 C and 3.3 ppm for 15 N) which is an indication for conformational heterogeneity of the Aβ molecules within the complex. In 2D 13 C-13 C correlation spectra ( Fig. 4 and Supplementary Fig. 13) 13 C side chain and backbone resonances can be identified for almost every amino acid residue type in the sequence. For several amino acid residue types, the number of distinct spin systems visible in the spectra is larger than the number of amino acid residues of this type in the amino acid sequence. For example, for Ala six spin systems have been found although the sequence of Aβ(1-42) only contains four Ala residues (Fig. 4). This means that not all Aβ molecules within the complex experience identical environments.  (Fig. 4) in α-helical, unstructured and β-strand like conformations a quantification was possible by integration of the peak regions (see Supplementary Fig. 18). Hence, these residues are predominantly in a β-strand conformation, except for Gly, which is a β-strand breaker.
Due to conformational heterogeneity, inhomogeneous line broadening and substantial resonance overlap in the 13 C-13 C and 15 N-13 C spectra, a full sequential resonance assignment for Aβoligo in complex with huPrP was not possible. However, from a series of PDSD spectra with different mixing times as well as 2D and 3D NCACX and NCOCX spectra it was possible to identify some interresidual correlations and to obtain site-specific assignments for some parts of Aβ in one predominant conformation (Supplementary Table 1). However, it is hard to tell whether all assigned resonances belong to one type of conformer, or to different conformers.
To elucidate whether the stoichiometry of Aβ and huPrP in the heteroassemblies has an influence on the conformations of Aβ molecules, we prepared and investigated a second sample, in which huPrP  was added in excess to 13 C, 15 N labeled Aβoligo. In this sample, all potential huPrP binding sites on Aβoligo should be occupied. Overall there is not much difference between sample huPrP(23-144)-Aβ* and huPrP(23-144)exc-Aβ* in a PDSD spectrum with a mixing time of 50 ms, except for minor changes (Supplementary Fig. 19).
As there are no major structural changes upon altering the huPrP concentration, we conclude that the conformational heterogeneity is not due to unoccupied huPrP binding sites in Aβoligo, but rather Aβoligo in complex with huPrP consists of inequivalent conformers and/or Aβoligo assemblies are different from each other.

Discussion
In this study we investigated the structures and the interaction of Aβ(1-42)oligo and huPrP by solid-state NMR spectroscopy (see Fig. 5 for a schematic representation of the structural features of the huPrP-Aβoligo complex). The N-terminal region of huPrP is rigid but has no regular secondary structure in the complex with Aβoligo. This is the case for both huPrP constructs, the full-length construct huPrP  and the N-terminal fragment huPrP . Minor structural changes to more α-helical like secondary structure are restricted to a region between A113 and L130, including the palindrome region. This palindrome region, known as the 'hydrophobic core', is highly conserved, and the AGAAAAGA region is highly amyloidogenic 44 . The palindrome segment has previously been suggested to be required for the attainment of the PrP Sc conformation and to facilitate the proper association of PrP Sc with PrP C to enable prion propagation 45 . Trapping the 'hydrophobic core' by binding to Aβ(1-42)oligo might explain the Aβ(1-42)-oligomer induced inhibition of prion propagation proposed by Sarell et al. 46 . In a recent study of a hydrogel-termed complex of full-length huPrP and Aβoligo 34 the formation of two additional α-helices, one in the octarepeat region (residues 51 to 91) and one in the palindrome segment (A 113 GAAAAGA 120 ), was postulated from the observation that chemical shifts observed for Gly and Ala are predominantly α-helical in their spectra 34 . Our results support the formation of the latter α-helix in the complex with full-length huPrP. Chemical shifts of Gly residues as well as all other N-terminal residues are predominantly random coil-like in the spectra (see For full-length huPrP in complex with Aoligo we observed some changes for Thr and Val residues from α-helical to random coil secondary chemical shifts compared to well-folded monomeric huPrP in solution 38 . huPrP was previously found to undergo a liquid-liquid phase separation in PBS buffer at pH 7.4 prior to complexation with Aoligo 34 . For pure liquid huPrP as well as for huPrP in a hydrogel with Aoligo chemical shift changes from α-helical to random coil values were described for Thr residues, which are mainly located in α-helices 2 and 3 34 . This observation was attributed to a loss of secondary structure during liquid-liquid phase separation and is still visible in the complex 34 . This fits to our observation that no cross correlation signals typical for Thr in α-helical conformation (from α-helix 2 and 3 in natively folded huPrP) were observed in our spectra.
Aβoligo in complex with huPrP consists of non-identical Aβ conformers. This is not surprising given the fact that the sample huPrP(23-144)-Aβ* contains eight times more Aβ (monomer equivalent) than huPrP(23-144) molecules. Not every monomer within the oligomer (containing 23 monomer units on average 47 ) might be able to bind to huPrP  in the same way and have therefore the same conformation 33 , as described above. These non-identical conformers can have different origins: (i) different types of monomers within the oligomer, because not every monomer can bind to huPrP (Aβ-huPrP vs. Aβ-Aβ interactions); (ii) polymorphism within the oligomer independent of the binding to huPrP (iii) polymorphism between different oligomers; or (iv) a combination thereof.
The secondary structure of Aβoligo in complex with huPrP shows a high degree of β-strand content. This could be due to fibrils that evolved over time. However, we took care not to obtain fibrils in our samples during preparation and there were no major shift changes in the CP and PDSD spectra in the following eleven months. This indicates that Aβoligos already contain Aβ monomer units that have at least in part the same secondary structure as in fibrils or protofibrils. This phenomenon has already been observed in early stage A oligomers 6,7 .
Assuming that the Aβoligo preparation yielded a heterogeneous collection of "small fibrils", of  11 with the PDSD spectrum of Aβoligo in complex with huPrP (Fig. 6). Nearly all predicted correlations from these different protofibril and fibril types are represented by correlation peaks in our oligomer spectra. These findings suggest that the Aβoligo preparation represents a heterogeneous mixture of -strand-rich assemblies, of which some may have the potential to evolve into the different fibril types. The conformational heterogeneity of Aβoligo is closely related to the polymorphism of Aβ fibrils and reflects the general propensity of Aβ to adopt variable β-structure conformers. (green ref. 36 and blue ref. 35 ) and an artificial protofibril (yellow) 11 . Separate overlays of this PDSD spectrum with spectra of these fibrils are shown in Supplementary Fig. 20 to 23.
The propensity of huPrP to efficiently bind to Aoligo and to "freeze" them in a non-dynamic and non-elongating state allowed us to investigate the conformers of Aoligo and the huPrP moiety by NMR over several months without noticeable changes in the sample. It is tempting to speculate whether this property of huPrP is a coincidence, or whether it is part of the longsought function of PrP. Regardless of whether PrP inhibits elongation of A oligomers and fibrils or whether PrP is a mediator of cytotoxicity of Aoligos, substances that compete with PrP for Aoligo binding and which thus can do the same job without the potential of mediating cytotoxicity may be of high therapeutic potential.

Preparation of high molecular weight hetero-assemblies from amyloid β oligomers and different human prion protein constructs in different molecular ratios
For sample preparation, Aβ(1-42) lyophilisates (either uniformly 13 C, 15  After addition of 0.03 % of sodium azide and incubation for 30 min the samples were centrifuged for two to five minutes at 16,100 x g, and the supernatant was removed. The sediment was washed twice with up to 2 ml of 30 mM Tris-HCl buffer, 0.03 % sodium azide, pH 7.4. After removal of the supernatant the samples were transferred into 3.2 mm MAS rotors with a Hamilton syringe and centrifuged. In total, four different samples were prepared in which either huPrP or A(1-42) was uniformly 13 C, 15 N labeled, using different huPrP constructs and molar ratios between huPrP and A(1-42) ( Table 1).

Characterization by density gradient ultracentrifugation, SDS-PAGE and RP-HPLC
For biophysical characterization of e. g. sample huPrP(23-144)-Aβ*, sucrose density gradient ultracentrifugation (DGC) was performed. To this end, 10 μl of the sedimented, but unwashed sample was diluted with 90 μl of 30 mM Tris-HCl buffer, pH 7.4 and applied on a discontinuous sucrose gradient (see 33 ) and centrifuged for 3 h at 259,000 x g and 4 °C. After fractionation, each of the 14 fractions was analyzed by Tris-Tricine SDS-PAGE and RP-HPLC as previously described 33 (Supplementary Fig. 3).

Preparation of solution NMR samples
For the sequence-specific backbone resonance assignments samples of 0.36 mM uniformly 13 C, 15

Solid-state NMR experiments
The Additionally, several multi-dimensional homo-and heteronuclear correlation experiments for the assignment were recorded. Experimental details of all spectra recorded are given in Supplementary Tables 2 to 6. For homonuclear 13 C-13 C spectra, proton driven spin diffusion (PDSD) 50 with mixing times between 10 to 300 ms was employed. Homonuclear double quantum correlation spectra were recorded with SPC5-recoupling 51 .
For site-specific assignment 15 N-13 C correlation spectra were recorded using SPECIFIC-CP 52 for frequency selective polarization transfer from 15 N to either 13 Cα or 13 CO and subsequent DARR-mixing. 2D NCA, NCACX and 3D NCACX and NCOCX spectra were used for the sequential walk through the backbone. During all acquisition and evolution times, high power broadband proton decoupling with SPINAL phase modulation 53 (radio frequency intensity between 71 and 91 kHz) was used. All spectra were processed with NMRPipe 54 with either squared and shifted sine bell or Gaussian window functions. The line width (FWHM) was estimated in 1D-slices (spectra processed with squared sine bell shifted by 0.35π or 0.40π) of 2D PDSD or NCACX/NCOCX spectra. 13 C chemical shifts were externally referenced with adamantane by setting the low frequency signal of adamantane to 31.4 ppm on the DSS reference scale. 15 N chemical shifts were indirectly referenced via the 13 C chemical shifts. All resonances were assigned in CCPN 55 . Integration of Aβ peaks was done in Topspin via the box sum method in a PDSD spectrum of huPrP(23-144)-Aβ*, measured at a temperature of ≈ 0 °C, a spinning frequency of 11 kHz and a mixing time of 50 ms.

Solution NMR experiments
For the sequence-specific backbone resonance assignments of uniformly 13 C, 15  To obtain sequence-specific backbone resonance assignments for huPrP

Competing interests:
The authors declare no competing financial interests.