The mechanism of Hsp90-induced oligomerizaton of Tau

RESULTS

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 (, ). 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 (, ) 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; (, )]: 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 (, ).

Hinderberger and co-workers (, ) 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 Δ _eff, which is the signal decay of the DEER time trace at time t = 3 μs ( Fig. 1C). While a DEER trace in the absence of Hsp90 delivers a reference Δ _eff value for each Tau sample, the change ΔΔ _eff upon addition of Hsp90 characterizes transitions in the conformational equilibrium: Negative ΔΔ _eff values indicate an increase in spin-spin separation, while positive Δ _eff values are consistent with the spins coming into closer proximity of each other (see details of modulation depth-based approach in fig. S7). This allows extracting distance information from DEER traces not analyzable in the conventional way.

The systematic analysis of the experimental Δ _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 (, ). On the one hand, these results demonstrate the capacity of the Δ _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 (, , , ).

It has been shown that Hsp90 induces oligomerization of Tau fragments (). 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 high-molecular 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 ().

To identify the oligomerization domain in Tau relevant for Hsp90-induced oligomerization, we performed intermolecular DEER measurements using singly spin-labeled Tau: Upon oligomerization, Δ _eff would increase locally where inter-Tau contacts are established. We observed very small Δ _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 Δ _eff for Tau-322* and Tau-354*, depicted as difference values ΔΔ _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 (, ). This is remarkable, as it suggests that the same stretch of Tau mediating fibril formation () is addressed by Hsp90 to promote the formation of oligomers.

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 τ _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 (). 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) (). The Tau/Hsp90 complex appears to be characterized by transient interactions between individual residues, involving a structural multiplicity of Tau.

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 (). 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 Δ _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). ΔΔ _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 (). 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.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaax6999/DC1

Supplementary Materials and Methods

Fig. S1. SDS gels of spin-labeled Tau.

Fig. S2. Mass spectrometry of spin-labeled Tau.

Fig. S3. Circular dichroism spectra of Tau.

Fig. S4. DEER data.

Fig. S5. RC model.

Fig. S6. Solvent-dependent RC model.

Fig. S7. Δ _eff as a measure for spin-spin separation.

Fig. S8. DEER data evaluation with different background corrections for doubly spin-labeled Tau.

Fig. S9. cw EPR data.

Fig. S10. QCM measurements.

Fig. S11. DEER data recorded in the presence of GdnHCl.

Table S1. Simulation of circular dichroism spectra.

Table S2. Kinetic analysis of QCM measurements.

Table S3. Overview of Δ _eff values and corresponding uncertainties (where applicable) as determined from various background analyses.