Friday, February 23, 2024

Local structural preferences in shaping tau amyloid polymorphism – Nature Communications

Mapping amyloid propensity within the tau RD

The structure and sequence composition of the amyloid core in different tauopathies is now well-established, with all known pathologic tau deposits containing repeats R3 and R4, whereas R2 is present or absent in different disease states14. Amyloid sequence prediction algorithms suggest that the different tau amyloid core sequences are not uniformly aggregation-prone. The sequence-based amyloid prediction algorithm WALTZ24 identifies two previously validated aggregation-prone regions, namely PHF6* in R2 (275VQIINK280) and PHF6 in R3 (306VQIVYK311)18,19 (Fig. 1b). However, WALTZ also predicts the existence of another amyloid motif, spanning residues 350VQSKIGSLDNITH362 (PAM4) in R4 of the tauRD domain, which has not been characterised previously (Fig. 1b, cyan area). To complement this analysis, we utilised CORDAX, a structure-based machine learning predictor that instead assesses the compatibility of the tauRD sequence with a cross-β structure25. CORDAX not only confirmed the predictions for PHF6*, PHF6, and PAM4 but also identified two additional segments, comprising 326GNIHHK331 and 344LDFKDR349 (Fig. 1b, purple line).

To determine experimentally the amyloid propensity of the entire tauRD domain, we designed a peptide library consisting of 41 peptides spanning its N- to C-terminus. This library was developed using a sliding window approach, generating 15-mer peptides with three-residue steps (Fig. 1a). For each peptide, we evaluated its amyloid-forming tendency using two distinct amyloid-reporting dyes, each with different charge:26 the positively charged Thioflavin-T (Th-T) and the negatively charged luminescent conjugated oligothiophene (LCO) pFTAA dye. In agreement with the WALTZ and CORDAX predictions, the results from the peptide screen confirmed the binding of both dyes to specific peptides within the tauRD (Fig. 1c, d). Two neighbouring peptides, each containing PHF6* in R2 (red-shaded area) and three adjacent peptides, harbouring PHF6 in R3 (green-shaded area), exhibited binding with both dyes, with one additional peptide containing PHF6 positively binding to pFTAA but not to Th-T. Notably, the peptide spanning residues 348–362, encompassing the predicted PAM4 in R4 (cyan-shaded area) exhibited strong binding for both dyes. Electron microscopy (EM) demonstrated that peptides containing PHF6*, PHF6, or PAM4 formed long, unbranched amyloid-like fibrils (Fig. 1e). Interestingly, neighbouring windows containing partial segments of the PAM4 motif did not exhibit any aggregation, suggesting that the intact motif is required for the formation of stable amyloid aggregates in isolation.

Two further segments were detected in the peptide screen. The first, which exhibited relatively weak binding to pFTAA, but no observable increase in ThT fluorescence, corresponds to the peptide spanning residues 318–332 (Fig. 1c, d, purple-shaded area). This region contains the 326GNIHHK331 sequence in R3 predicted by CORDAX. Negative stain EM showed that this peptide formed a dense network of thin amyloid-like aggregates (Fig. 1e, purple box). The second additional segment consists of the peptide spanning residues 333–347 in R4, which exclusively bound to Th-T (Fig. 1c, d, yellow-shaded area) and formed unbranched amyloid fibrils (Fig. 1e, yellow box). This peptide contains the previously predicted 337VEVKSE342 motif27, as well as the 344LDFKDR349 sequence predicted by CORDAX (Fig. 1a). Our peptide screen thus confirms that the amyloidogenic nature of the tauRD sequence is not uniform. Instead, specific segments demonstrate an increased inherent propensity to form cross-β amyloid structure, while others clearly require additional structural context to be incorporated into the amyloid core observed for these regions in pathogenic tau fibril isolates.

PAM4 forms amyloid fibrils that promote cellular tau seeding

To perform a deeper analysis of the amyloidogenic properties of the identified PAM4 region, we next synthesised a high purity (>99%) peptide corresponding to this segment (matching residues 350–362, Supplementary Fig. 1a, b) and characterised its ability to assemble into amyloid fibrils in more detail. The kinetics of amyloid assembly monitored using Th-T fluorescence revealed that PAM4 forms amyloid fibrils. End-state fluorescence values reveal that Th-T is bound in a concentration-dependent manner, with PAM4 showing similar Th-T levels at a matching concentration (200 μM) compared to the 348–362 peptide identified by our tau screening assay (Fig. 2a). X-ray diffraction patterns produced from oriented fibrils of the peptide were indicative of a cross-β architecture, with a meridionally oriented reflection at 4.7 Å and an equatorial reflection at 10.9 Å, corresponding to the stacking and packing distances of β-strands and β-sheets along the fibril axis, respectively (Fig. 2b). FTIR spectroscopy revealed two prominent peaks at 1631 cm−1 and 1680 cm−1 within the amide I region, both indicative of a β-sheet-rich conformation (Fig. 2c). Thin films containing end-state aggregates were positively stained with the Congo red dye, as seen under bright-field illumination, and exhibited green birefringence that is typical of amyloid deposits when viewed under polarised light (Fig. 2d). Atomic force microscopy (AFM) and transmission electron microscopy (TEM) revealed the formation of long, unbranched amyloid-like fibrils (Fig. 2e, f), with a higher level of polymorphism compared to the 348–362 peptide, as we could observe straight and laterally interacting fibrils, as well as twisted helical and ribbon-like morphologies (Fig. 2f).

Fig. 2: Biophysical characterisation of the amyloid-like properties of PAM4.

a Concentration-dependent Th-T kinetic assays of the PAM4 peptide (n = 3 independent repeats), along with end-state Th-T fluorescence values shown in the right bar plot, with individual values shown as points. b Cross-β diffraction pattern produced by oriented fibres containing PAM4 peptide fibrils. Intensity quantification along the meridional and equatorial axis of the pattern indicate the presence of an intense 4.7 Å and 10.9 Å reflection, respectively. c FTIR spectrum produced from fibril deposits of the PAM4 peptide. The prominent 1631 cm−1 and 1680 cm−1 peaks are indicative of a dominant β-sheet secondary structure. d Polarised microscopy reveals an apple-green birefringence shown by PAM4 peptide deposits which typically signifies the presence of amyloid aggregates. (Scale Bar = 100 μM). e Atomic force microscopy imaging of PAM4 fibrils. Multiple helical morphologies can be observed in a single field of view. A single representative image is shown (n = 3 independent repeats). f Electron micrograph of fibrils formed by assembly of the PAM4 peptide. Higher magnifications of individual fibrils (lower images) showcase the presence of highly polymorphic amyloid fibrils. A single representative image is shown (n = 3 independent repeats). gh Concentration-dependent seeding quantification performed by counting the percentage of expressing cells containing fluorescent puncta (n = 3 independent repeats) and representative images of the dose-dependent seeding of tauRD-YFP conjugate construct with PAM4. (Scale bar = 20 μm). i, j Representative images (Bar = 20 μm) and quantification of puncta-positive cells transiently expressing the tauRD-YFP construct, following transduction with 5 μM of peptide seeds. The individual points are colour-coded to match the regions highlighted in Fig. 1, with underlined segments representing the aggregation motifs predicted by Cordax and Waltz. Bar plots represent mean values ± SEM (n = 4 independent samples). The solid and dashed horizontal lines indicate the mean and SEM corresponding to the untreated biosensor condition. Statistical significance was determined using one-way ANOVA with Dunnett’s correction for multiple comparisons.

We also investigated the amyloid nucleating properties of the same segment against the intact tauRD sequence within a cellular context. To do this, we generated seeds by sonicating mature PAM4 peptide amyloid fibrils and adding these to HEK-293 cells transiently expressing tauRD conjugated to YFP (Methods). By counting the number of cells containing yellow puncta using automated image analysis (Methods), the derived dose-response curve revealed a strong seeding efficiency of the PAM4 seeds (calculated EC50 = 4.1 μM) (Fig. 2g, h). In fact, when comparing the seeding capacity of PAM4 with other amyloidogenic peptides from the tauRD identified in our screen, we found that the PAM4 peptide exhibits seeding potency 10- to 100-times higher than any of the other peptides, suggesting that it is one of the strongest tauRD nucleating regions (Fig. 2i, j).

PAM4 modulates cellular seeding of AD and other primary tauopathy-derived seeds

We next questioned the role of PAM4 in the seeding efficiency of the tauRD within a cellular context and compared its effects to the previously established PHF6* and PHF6 aggregation motifs. To do so, we transiently expressed YFP-tauRD conjugates (incorporating the P301S mutation) lacking either 275VQIINK280 (ΔPHF6*), 306VQIVYK311 (ΔPHF6) or 350VQSKIGSLDNITH362 (ΔPAM4) motifs in HEK293 cells and compared their induced intracellular aggregation to that of the full-length tauRD (P301S) construct. To induce aggregation, we used seeds derived from heparin-induced recombinant tau fibrils (rTau) formed in vitro (Fig. 3a), as well as seeds formed from tau extracted from the brains of patients diagnosed with AD (Supplementary Table 1). Dose-response curves of seeding efficiency, reported as the number of expressing cells containing fluorescent puncta, showed that tau seeding was abolished in ΔPHF6 expressing cells, regardless of the source of the seed aggregates (Fig. 3b, c, purple curves). This finding corroborates the crucial role of PHF6 in tau amyloid aggregation both in vitro and in cells18,19. We found that the PHF6* motif contributes to the seeding of in vitro generated tau seeds, but not for the seeds from AD patients. Specifically, a significant reduction in seeding efficiency was observed in ΔPHF6*-expressing cells when treated with rTau seeds (Fig. 3b, c, red curves), resulting in a 5-fold decrease of the EC50 value of ΔPHF6* cells when compared to full-length tauRD (P301S) (Fig. 3d).

Fig. 3: Cellular screening using deletion constructs of the tauRD reveals a differential relationship of APRs to tau aggregate strains.
figure 3

a Intracellular seeding of cells expressing tauRD-YFP is reported by counting the number of cells with a punctuate morphology in a concentration-dependent manner upon treatment with rTau seeds. (Scale bar = 20 μm). b Dose-response curves after the treatment of cells expressing tauRD (WT) ΔPHF6*, ΔPHF6 or ΔPAM4 with various concentrations of rTau seeds or seeded with extracts isolated from three independent AD cases (n = 6, three independent samples with two technical repeats for each case). Individual points presented as mean values with ±SD. c Representative images of treated cells with selected seed concentrations (shown in arrow in b, Scale bar = 20 μm). d Inverted effects of the ΔPAM4 and ΔPHF6* on seeding efficiencies, shown as changes in EC50 values, of recombinantly produced or AD extracted tau aggregates validates indicate that a bias towards specific tau polymorphs, in contrast to ΔPHF6 that is generally critical for tau aggregation. Bar plots represent mean values ± SD (n = 3 independent samples).

No change in seeding efficiency was observed when cells expressing the ΔPAM4 construct were treated with heparin-prepared seeds (Fig. 3c, d, blue triangles), suggesting that PAM4 does not contribute to this tau polymorph. However, a notable decrease in seeding efficiency was observed in these cells for seeds extracted from three independent AD patients (AD1-3) (Fig. 3b–d), where seeding potential was much higher with EC50 values in the fM range (Fig. 3d). In all cases, deletion of PAM4 significantly impaired the ability of AD extracts to induce seeding in a cellular context, with more than a 1000-fold decrease for ΔPAM4 cells compared with cells expressing the intact tauRD (measured by their EC50 values) (Fig. 3d). Contrastingly, seeding efficiency with AD extracts was unaffected upon deletion of PHF6*, demonstrating that this region is not important to seed AD polymorphs.

Previous structural studies have shown that R4 of tauRD (which contains PAM4) is an integral part of AD protofilaments28, but is absent from the ordered cores of heparin-induced recombinant tau fibrils29, while PHF6* shows the opposite organisation (present in the ordered core of heparin-induced recombinant tau fibrils). This suggests that the seeding efficacy differences observed above could potentially be explained simply by considering which segment is ordered in the different fibril structures. To investigate if this is the case, we generated cells that express tauRD in which the 337VEVKSE342 or 343LDFKDR349 segments are deleted, both sequences that correspond to elements identified by our aggregation screen (Fig. 1) or by previous reports30. Although both segments are integral parts of the structural core of AD-derived tau fibrils, these deletion constructs seeded with equal efficiency to that of the cells expressing the intact tauRD (Supplementary Fig. 2), confirming the integral role of PAM4 for templating by AD-patient tau seeds.

Next, we sought to investigate if PAM4 further modulates the seeding of polymorphs derived from other representative 3R, 4R, and 3R/4R tauopathies. Parallel cellular assays for extracts derived from a CBD, PSP, PiD and a fourth independent AD (AD4) patient case (Supplementary Table 1) once more validated the known importance of PHF6, as the ΔPHF6 deletion resulted in a ubiquitous loss of cellular seeding regardless of the source of the tau seeds (Supplementary Fig. 3). PAM4 deletion impairs cellular seeding induced by AD (Supplementary Fig. 3a), PSP extracts (Supplementary Fig. 3b), and to a lesser extent CBD-derived extracts (Supplementary Fig. 3c). Except for PHF6, none of the other motif deletions impede cellular seeding induced by PiD-extracted aggregates compared with cells expressing the intact tauRD (Supplementary Fig. 3d). PHF6* deletion resulted in slightly increased seeding induced by PiD compared to the intact tauRD. In line with recent evidence31, this could potentially be explained as the result of shedding segments of the fuzzy coat region, as the R2 domain is not incorporated in the core of PiD polymorphs. In conclusion, these assays demonstrate that cellular seeding propensity differs between ex vivo tau polymorphs and highlight a key role for the PAM4 segment, particularly for AD-, CBD-, and PSP-derived tau aggregates.

Cryo-EM determination of PAM4 polymorphic protofilament folds

The structure of amyloid fibrils formed by the PAM4 peptide was explored using cryo-EM. The polymorphic propensity of PAM4, consistent with negative stain and AFM data (Fig. 2e, f), was validated by the appearance of multiple fibril morphologies in the micrographs and after 2D classification of the data (Supplementary Fig. 4a, b). Specifically, we were able to identify 2D-classes containing potential two (65%), three (28%) and four (10%) protofilament polymorphs assembled from conserved core folds (Supplementary Fig. 4c–e). The structure of the two-protofilament morphology (PAM4 Type 1) was determined at 2.6 Å resolution (Fig. 4a–c & Supplementary Fig. S5), with a helical twist of 359.32° and rise of 4.86 Å corresponding to a crossover of 125 nm (Supplementary Table 2). Each protofilament contained six PAM4 subunits, giving a total of twelve subunits per layer of the fibril with each protofilament comprising a head-to-tail ring of PAM4 peptides (Fig. 4b, c). The lower abundance, larger fibril morphologies of the PAM4 peptide fibrils could not be resolved to high resolution but appeared to contain the same protofilament building blocks as PAM4 Type 1 with increasing assembly sizes (Supplementary Figs. 4 and S5).

Fig. 4: Cryo-EM structures of PAM4 fibrils show a diversity of folds.
figure 4

a Slices through the cryo-EM map of each PAM4 fibril structure, made by averaging the central 6x slices of the post-processed, sharpened map to display approximately a single helical layer. Scale bar = 3 nm. b Cryo-EM maps (grey, transparent surface) with fitted atomic models for each solved structure, displayed in the same order as in a. Each peptide chain is coloured blue-to-red from N- to C-terminus and a single helical layer is shown. c PAM4 structures represented as cartoon loops, coloured by subunit fold as indicated in d and with the N-terminal acetyl-Val or Fmoc-Val and C-terminal amide-His residues shown as sticks and labelled. Structures are displayed in the same order as in a. d Schematic representation of individual residues for the distinct monomeric conformations adopted by PAM4 and identified by cryo-EM. e Close-up views of the minimal repeating unit in each structure, displayed with cartoon backbone and stick side chains coloured by subunit as in c. Inter-protofilament steric zipper interactions are highlighted with shaded backgrounds, with arrows indicating the location of the GS-bend formed by Gly355 and Ser356. The insert highlights the formation of an intramolecular salt bridge between Lys353 and Asp358 that further stabilises the monomeric PAM4 FoldC.

We also determined the structure of PAM4 amyloid fibrils formed by a lower purity (92%) peptide batch (Supplementary Fig. 1c, d). Two-dimensional (2D) classification revealed that this peptide batch also formed multiple fibril polymorphs (Supplementary Fig. 6), with three distinct PAM4 fibril structures that could be determined to high resolution (Fig. 4a–c & Supplementary Fig. S7). The most abundant polymorph, PAM4 Type 2 (representing 28% of the total population), was resolved to a resolution of 2.8 Å (Fig. 4a–c & Supplementary Fig. S7). This polymorph has six peptide subunits per layer with a helical twist and rise of 358.55° and 4.8 Å, respectively, corresponding to a crossover distance of 60 nm (Supplementary Table 2). The remaining two PAM4 fibril structures were determined at resolutions of 2.7 Å (Type 3, 15% of the total population) and 2.9 Å (Type 4, 13% of the total population), respectively (Fig. 4a–c & Supplementary Fig. S7). The Type 3 and Type 4 fibrils share similar structural properties including crossovers around 80 nm (Supplementary Table 2) and containing eight PAM4 subunits per layer of the fibril core, differing only in a slight re-positioning of the inter-protofilament interface. The resolution of the maps, in combination with liquid chromatography-mass spectrometry (LCMS) data, facilitated the identification of an adduct coming from a minor population of the peptide containing an N-terminal Fmoc protection group from peptide synthesis (~7.4%, Supplementary Fig. 1). It is not clear what percentage of the peptide in the total population of fibrils contained this protected peptide, but in the final selected fibril segments that yielded high-resolution maps, the occupancy was high. Interestingly, when compared to N-terminal acetylation, Fmoc protection has been previously suggested to promote the formation of hydrogels by providing additional π-π stacking interactions32. Similarly, stacked protected N-termini pack at the protofibril interfaces of each of the three fibril structures with the C-termini extending towards the anterior fibril surface (Fig. 4c), which could potentially explain the additional structural morphologies resolved in the presence of the Fmoc protection group.

All four of the PAM4 fibril structures are inferred to be left-handed from the resolution of the cryoEM maps (Supplementary Fig. 8a), as well as from cryo-electron tomography (cryoET) reconstructions of Type 1 fibrils (Supplementary Fig. 8b, c). Altogether, PAM4 peptide monomers exhibited four unique conformations (named here Folds A-D) within the four solved fibril structures (Fig. 4d, e). Interestingly, the folds were interchangeable within the different fibril types, with Type 1 contaning FoldA and B, Type 2 fibrils containing FoldB, C and D, and Type 3 and Type 4 fibrils containing FoldC and D (Fig. 4c, d). In all cases, the folds share a C-terminal β-strand (residues 358–362) but differ in the way they orientate the N-terminus about an intermediary GS-bend (residues 355–356, Fig. 4d, e). Additionally, FoldC contains an intra-subunit salt bridge between K353 and D358 that stabilises its highly kinked (>90°) conformation (Fig. 4e). Importantly, all of the complex six-to-twelve subunit fibril layers in the PAM4 Type 1–4 polymorphs are built from repeating units of three different arrangements of the four peptide folds, which form either face-to-back or back-to-back steric zippers between their C-terminal β-strands (Fig. 4e). In the FoldA-FoldB building block (seen in PAM4 Type 1 fibrils), the FoldA subunits are similar to FoldB but bend further around the GS region to cap the end of the adjacent peptide chain (Fig. 4c, e).

PAM4 conformations are representative of disease-associated tau polymorphs

Despite the fact that the four PAM4 amyloid peptide folds were obtained through self-assembly of synthetic peptide derivatives, superimposition onto the structures of full-length human-derived tau amyloid fibril folds revealed that the structural polymorphism of the PAM4 motif in isolation matches its conformations within the protofilaments of every major class of human tauopathies, including 3R, 4R and 3R/4R tau isoforms (Fig. 5). Specifically, the FoldC conformation of PAM4 stabilised by the K353-D358 salt-bridge is also found in tau C-fold protofilament polymorphs linked to Alzheimer’s disease (AD), Gerstmann-Sträussler-Scheinker (GSS), primary age-related tauopathy (PART) and cerebral amyloid angiopathy (CAA), while showing reasonable fit to chronic traumatic encephalopathy (CTE) polymorphs (Fig. 5). FoldB PAM4, which has a flipped N-terminal compared to the previous fold, perfectly matches the 4R PSP-derived tau fibril cores and shows a C-terminal mismatch to the 3R PiD tau polymorph (Fig. 5). Finally, FoldD PAM4 matches R2:R3:R4 (4R) tau strains that are associated with corticobasal degeneration (CBD) and argyrophilic grain disease (AGD) (Fig. 5). In conjunction with our cell assays showing that PAM4 reduced AD, PSP and CBD seeding, our findings here show that the cryo-EM structures of PAM4 amyloid polymorphs in isolation are representative of the same patient-derived tau polymorphs.

Fig. 5: Polymorphism of ex vivo derived tau fibrils traces back to the innate structural features of PAM4 fibrils.
figure 5

The PAM4 folds recovered in isolation match its conformation in patient-derived tau amyloid fibril structures belonging to each branch of their previously proposed structural classification14, including 3R, 4R and 3R/4R isoforms. Structural alignments reveal that the PAM4 FoldC matches the AD conformation and shows reasonable fit to CTE (3R/4R), as shown by their respective RMSD calculations (1.43 Å and 1.63 Å, respectively). Similarly, FoldB matches the PSP conformation and shows a mismatch at the C-terminal of the region in the PiD conformation, respectively (RMSD values of 0.53 Å and 1.69 Å). Finally, FoldD perfectly overlaps the CBD and AGD (4 R) conformations (RMSD values of 1.19 Å and 1.06 Å, respectively). Zoom-in inlets highlight the superposed PAM4 segments from tau polymorphs and the individual folds observed in isolation using cryoEM. Tau structures are coloured in grey, whereas individual PAM4 folds are coloured as in Fig. 4d.

PAM4 and other motifs stabilise polymorphic tau fibril cores

We calculated and compared the per residue energy profiles of the amyloid core sequence in structurally categorised tau polymorphs (Fig. 6a–c). The cumulative per-residue energy profiles calculated for all tau fibril polymorphs indicate that specific regions play a dominant role in determining the stability of amyloid cores across different polymorphs, with other regions having relatively little stabilising influence (Fig. 6a, b). For example, PHF6* in R2, PHF6 in R3 and PAM4 in R4 consistently display stabilising effects whereas, for instance, residues 321–325 show consistent destabilization of the fibril core structure (Fig. 6b).

Fig. 6: Thermodynamic analysis of tau fibril polymorphs.
figure 6

a Mapping the positions of identified aggregation motifs on tau fibrils polymorphs of major structural classes. b, c Heatmap plot indicating the per residue energy contributions for structurally determined tau amyloid fibril cores derived from different patient extracts or fibrils produced in vitro45. PDB IDs of individual tau fibril structures are shown on the Y-axis, separated by dashed lines based on disease type, whereas the X-axis indicates tau residue numbers. The top panel bar plot (b) indicates the sum of energy contributions per residue for all structures shown in the heatmap, with the identified aggregation motif regions shown in shaded boxes and coloured as in a. dh Tau fibril polymorphs are stabilised primarily by the identified aggregation motifs. Residue sidechains are shown in ball representation, with important aggregation motif interactions highlighted and the rest of the residue sidechains faded out. Residues are coloured based on the calculated energies shown in c.

An examination of individual energy profiles confirms that within the context of full-length tau fibril cores and consistent with previous findings33, PHF6 emerges as the most strong amyloid core stabilising segment across various tau structures, both in in vitro and ex vivo polymorphs (Fig. 6c). Additionally, the previously identified amyloidogenic segment PHF6* is also a key contributor to the stability of tau polymorphs in which it is present. This includes primarily in vitro-formed polymorphs and R4 polymorphs with longer protofilament cores, such as CBD/AGD and PSP/GPT/GGT cores. Although PHF6* and PHF6 are the most sSupplementary Table tructural segments in R2 and R3, respectively, this analysis also demonstrates that PAM4 emerges as the most sSupplementary Table egment of R4, and the second most stabilising region in total, in tau fibril cores polymorphs (Fig. 6b, c).

Tau polymorphism arises from amyloid motif rearrangement into tertiary folds

The fact that the local architecture of PAM4 amyloid folds is not overruled by tertiary interactions in full-length tau amyloid cores suggests that the intrinsic structural propensity of PAM4 restricts the available conformational freedom of the tau protofilament. The way in which the PAM4 conformations are incorporated in fibril polymorphs is in good agreement with the classification recently proposed for tau14 (Figs. 5 and 6a). R3:R4-containing polymorphs, including AD, PART and CTE protofilament folds, are stabilised by the PAM4 FoldC conformation. This L-shaped conformation of PAM4 stabilizes one leg of the horseshoe structure of these polymorphs, forming a heterotypic interface with 326GNIHHK331, while PHF6 stabilizes the other leg (Fig. 6d). Interestingly, thermodynamic analysis of this PAM4 conformation in the full-length C-fold protofilaments reveals that the C-terminal segment forming this heterotypic interface with GNIHHK is the primary source of stability, with the less favourable N-terminal segment of this conformation being stabilised primarily by the formation of the K353-D358 salt-bridge, respectively (Fig. 6d).

The R1:R3:R4-containing (3R) structure of PiD also adopts a two-legged – albeit less curved – protofilament fold. In this conformation, PAM4 stabilises one leg, whereas a heterotypic interface formed between the 337VEVKSE342 aggregation motif and the extremely stable PHF6 motif stabilizes the other, as also shown in previous studies27 (Fig. 6e). The R2:R3:R4-containing (4R) tau polymorphs display a mixture of protofilament folds containing diverse conformations of the PAM4 motif. The AGD and CBD protofilament folds incorporate a longer section of the tauRD but adopt a more compact structure. These cores are stabilised centrally by a heterotypic PHF6-VEVKSE steric zipper, just as in the case of the PiD polymorph. The FoldD conformation of PAM4 stabilises the R4 segment which wraps around the core formed by the heterotypic zipper, with the N-terminal PHF6* closing the structure and acting as an additional molecular staple by interacting with the destabilised C-terminus of the amyloid core (Fig. 6f). In the amyloid core of fibrils derived from PSP patients, the FoldB conformation of PAM4 forms a partial heterotypic steric zipper interface of exceptional stability with PHF6 that staples one side of the elongated fibril core, whereas the other side is stabilised through interdigitation of the PHF6*, 326GNIHHK331 and 337VEVKSE342 amyloid motifs, respectively (Fig. 6g). Finally, alternative interfaces are formed between PAM4 and PHF6 in GPT and GTT polymorphs, stabilising a particularly unstable PAM4 conformation in the latter (Fig. 6h), which may potentially explain why this PAM4 architecture was not recovered in isolation. Together, these residue energy profiles reveal that, despite their structural diversity, tau fibril cores exhibit significant similarities in terms of the structural elements governing their thermodynamic stability and that only a few specific amyloidogenic segments, including PAM4, serve as common building blocks.

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