Tau is an intrinsically disordered protein (IDP) known to bind to and stabilize microtubules (MTs) and regulate axonal transport in its physiological function (13). In pathology, filamentous aggregates of Tau constitute a hallmark of neurodegenerative diseases, among them Alzheimers disease (AD) (4). Both MT binding and self-aggregation of Tau are mediated by the Tau repeat domain (Tau-RD) consisting of four imperfect repeats in the longest Tau isoform (Fig. 1A) (5, 6).

While Tau in solution is generally disordered and highly dynamic, long-range interactions mediate folding back of both termini onto Tau-RD, resulting in an overall paper-clip arrangement of monomeric Tau, which has been well established by a variety of experimental techniques such as nuclear magnetic resonance (NMR) and fluorescence resonance energy transfer (FRET) (7, 8). In filamentous aggregates of Tau, Tau-RD forms the ordered filamental core, while N- and C-terminal regions remain a disordered fuzzy coat (9, 10). Tau filaments exist in different morphologies with notable differences in the fold of the filamental cores, which are probably disease specific (11, 12). Although fibrils have long been considered the neurotoxic species, neuronal death appears rather to be caused by prefibrillar soluble aggregates and oligomers of Tau (13, 14), which are also considered responsible for spreading Tau pathogenicity from cell to cell in a prion-like concept (15, 16).

The molecular chaperone heat shock protein 90 (Hsp90) (17, 18) initiates proteasomal degradation (1921) and induces oligomerization of Tau (2224). Tau-RD is part of the Hsp90/Tau interaction interface (25). While insight into the molecular mechanism of the emergence of toxic Tau oligomers is highly relevant in the context of neuropathology, its structural principle is elusive.

The lack of a defined three-dimensional fold of IDPs like Tau makes their structural characterization challenging. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling has proven powerful in the investigation of IDPs and their aggregation behavior also in the presence of diverse interaction partners (2631). EPR spectroscopy (i) provides information about the side-chain dynamics of a single residue (32). Dipolar spectroscopy, i.e., DEER (double electron-electron resonance) spectroscopy, (ii) gives access to distance information in the nanometer range between two spin labels by measuring their magnetic dipolar interaction frequency dd (3335). Here, we exploit the combination of these approaches to investigating the molecular mechanism of Hsp90-induced Tau oligomerization.

We genetically engineered Tau derivatives containing one or two cysteines at specific sites and performed thiol-specific spin labeling (Fig. 1B). A range of biochemical and biophysical assays was used to monitor the success of the labeling reaction and the structural integrity of the protein (figs. S1 to S3 and table S1).

Next, we set out to characterize the structural properties of Tau by obtaining long-range intramolecular distance information with DEER on doubly spin-labeled Tau. Typical experimental DEER form factors for Tau are shown in Fig. 1C (full data in fig. S4) in comparison to simulated data for a hypothetical, well-defined distance. In contrast to the latter, the experimental traces for Tau showed no distinct modulations, indicating a broad distribution of spin-spin distances and thus implying a vast conformational ensemble of Tau in solution.

For these experimental DEER traces, the standard method for DEER data analysis fails, and the extraction of precise distance distributions is precluded (36, 37). First, we tested whether the experimental DEER data are in agreement with a simple random coil (RC) model (fig. S5). We chose RC model parameters as published by Rhoades and co-workers (38, 39) for assessing the results of FRET experiments on Tau. For certain spin-labeled stretches of Tau, e.g., Tau-17*-103*, the RC model agreed well with the experimental results (Fig. 1D and fig. S5), indicating an RC-like structural ensemble in the corresponding Tau segments. However, the RC model cannot describe the whole DEER dataset even taking variation of RC parameters depending on solvent quality into account [see fig. S6; (40, 41)]: For Tau-17*-291* and Tau-17*-433*, the deviation between the experiment and the RC model indicates a considerable contribution from Tau conformations more compact than RC (Fig. 1E and fig. S5). This is in good agreement with the well-established finding that Tau does not adopt RC conformation in solution but rather a paper clip (7, 8).

Hinderberger and co-workers (36, 37) proposed a data analysis procedure, which we adapted for analyzing the broad conformational ensemble of a large IDP like Tau. We evaluated the DEER data using the effective modulation depth eff, which is the signal decay of the DEER time trace at time t = 3 s (Fig. 1C). While a DEER trace in the absence of Hsp90 delivers a reference eff value for each Tau sample, the change eff upon addition of Hsp90 characterizes transitions in the conformational equilibrium: Negative eff values indicate an increase in spin-spin separation, while positive eff values are consistent with the spins coming into closer proximity of each other (see details of modulation depthbased approach in fig. S7). This allows extracting distance information from DEER traces not analyzable in the conventional way.

The systematic analysis of the experimental eff values supports the paper-clip model proposed on the basis of FRET and NMR experiments for Tau in solution, where N and C termini are in proximity to each other, and Tau-RD is in an overall more compact fold than RC (7, 8). On the one hand, these results demonstrate the capacity of the eff approach for obtaining structural information from DEER traces reflecting vast protein ensembles, while on the other hand, they define the paper clip as a reference structural ensemble of Tau in solution, which is in agreement with the results obtained for the Tau structural ensemble in previous studies, suggesting a paper clip or S shape in solution (7, 8, 39, 42).

It has been shown that Hsp90 induces oligomerization of Tau fragments (22). Here, we analyzed the oligomerization behavior of full-length Tau by density gradient centrifugation (Fig. 2). Pure Tau was mainly found in its monomeric form, while heparin induced the formation of mature fibrils. In the presence of Hsp90, the amount of small oligomeric Tau species increased. Notably, the formation of highmolecular weight Tau aggregates and fibrils was prevented in the presence of Hsp90. Electron micrographs of K18 Tau fragments in the presence of Hsp90 also show the formation of small protein conglomerates, while fibril formation is prevented (43).

Dot blot summarizing the results of density gradient centrifugation and quantification (the color code represents Tau preparations as reported on top of the right graph): Pure Tau is mostly monomeric. Heparin induces formation of highmolecular weight fibrils. Hsp90 leads to an increase in small Tau oligomeric species, while formation of fibrils is prohibited. A.U., arbitrary units.

To identify the oligomerization domain in Tau relevant for Hsp90-induced oligomerization, we performed intermolecular DEER measurements using singly spin-labeled Tau: Upon oligomerization, eff would increase locally where inter-Tau contacts are established. We observed very small eff values for all Tau derivatives in the absence of Hsp90 (Fig. 3), indicating only minor subpopulations of oligomeric Tau species. Addition of Hsp90 leads to a considerable increase in eff for Tau-322* and Tau-354*, depicted as difference values eff. This suggests that the oligomerization interface is located in Tau-RD and specifically in R3/R4. Notably, Tau oligomerization initiates in the same Tau region responsible for AD fibril formation and Hsp90 binding (11, 25). This is remarkable, as it suggests that the same stretch of Tau mediating fibril formation (25) is addressed by Hsp90 to promote the formation of oligomers.

Information about intermolecular Tau/Tau interactions obtained with DEER of singly spin-labeled Tau in the absence (dark gray) and presence (light gray) of Hsp90. Nonzero eff values represent small amounts of nonmonomeric Tau in the absence of Hsp90. Hsp90 increased eff values for Tau-322* and Tau-354* (light green bars) in accordance with an increase in Tau oligomers mediated by R3/R4 of Tau-RD. Positions probed in the experiment are also indicated on a schematic representation of the Tau sequence, with indicated Hsp90-binding site (25) and the core of the AD fibril (11), yellow and green stars indicating spin labeling positions without and with changes in eff upon addition of Hsp90, respectively.

The dynamic properties of Tau in solution and with Hsp90 are reported by EPR spectra of spin-labeled Tau side chains. In general, we observed rather fast rotational dynamics with rotational correlation times corr around 1 ns (Fig. 4A). This is in accordance with Tau presiding in a largely unstructured state with a broad conformational ensemble and a high degree of dynamical disorder (26). Addition of Hsp90 induced only subtle changes in the spectra (fig. S9), indicating that dynamic disorder in Tau persists also when bound. The generally still fast dynamics in the Tau spectra hints toward a transient nature of the Tau/Hsp90 complex, as only a small portion of spin-labeled Tau might be motionally restricted by intermolecular contacts, while other Tau molecules retain unrestricted rotational diffusion. We determined the half lifetime of the Tau/Hsp90 complex by quartz crystal microbalance (QCM) affinity measurements at ~10 s, which is typical for transient protein-protein interactions (fig. S10 and table S2) (44). The Tau/Hsp90 complex appears to be characterized by transient interactions between individual residues, involving a structural multiplicity of Tau.

(A) Local side-chain dynamics accessed by cw EPR of 28 M singly spin-labeled Tau derivatives: Rotational correlation times corr determined in the absence (dark gray) and presence (light gray) of 56 M Hsp90 and respective changes corr (purple/pink) are shown. Arrows indicate a decrease (pink) or increase (purple) in side-chain mobility at the respective site. (B) Intramolecular distance information obtained by DEER spectroscopy with doubly spin-labeled Tau derivatives: Effective modulation depths eff determined in the absence (dark gray) and presence (light gray) of Hsp90 and respective changes eff (light/dark green) are shown. Arrows indicate a decrease (light green) or increase (dark green) in spin label separation.

We observed local restrictions of the reorientational mobility for spin-labeled side chains Tau-291* and Tau-322* in the presence of Hsp90. Both residues are located in Tau-RD, which has been identified as the Hsp90-binding region before (25). Thus, the altered dynamics are attributed to direct Tau/Hsp90 interaction, while also oligomer formation might restrict side chain dynamics of Tau-322*.

Spin label mobilities increased in Tau-17* and Tau-103* upon addition of Hsp90, indicating that these side chains gain a larger conformational space. Thus, one might speculate that the N terminus detaches from Tau-RD upon binding of Hsp90, opening up the paper-clip fold.

To elucidate the structural influence of Hsp90 on the Tau conformational ensemble, we performed DEER spectroscopy of doubly spin-labeled Tau. DEER traces remained modulation free upon addition of Hsp90 (fig. S4). Thus, dynamic disorder prevails in Tau also when interacting with the chaperone (Fig. 1C). Addition of Hsp90 changed eff values, indicating a shift in the conformational equilibrium of Tau (Fig. 4B): A pronounced increase in the average spin-spin separation occurred for Tau-17*-291* and Tau-17*-433*. This indicates that the N terminus detaches from both Tau-RD and the C terminus and folds outward, opening up the paper clip (Fig. 5). eff values suggested a slight stretching of N-terminal Tau between Tau-17*-103* and of Tau-RD in the region between Tau-291*-322* in R2/R3, while the overall dimension of Tau-RD between Tau-244*-354* remained unchanged. While individual repeat sequences, e.g., R2/R3 expanded while accommodating Hsp90, there seems to be considerable flexibility in the remaining Tau-RD for preserving its overall dimension. A similar structural reorganization of Tau toward an open conformation was reported upon binding to tubulin, where stretches between individual repeats expanded, while the overall dimension of Tau-RD remained unaffected (38). Our results report the conformational basis of Tau oligomerization in the presence of Hsp90 and suggest that binding to Hsp90 opens the compact Tau solution structure, exposing Tau-RD residues and presenting them to other Tau molecules. As the Tau/Hsp90 complex is of a transient nature, oligomerization of Tau molecules may then occur via exposed Tau-RD.

A structural model of Tau in the absence (left) and presence of Hsp90 (right) can be derived from EPR data of the Tau conformational ensemble. In the absence of Hsp90, Tau adopts a paper-clip shape with both termini folded back onto Tau-RD. In the presence of Hsp90 the N terminus folds outward, thereby uncovering Tau-RD.

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