Fig. 4.

Phosphorylation-dependent complexation of Tau. a Binding affinity of Tau to Hsp90 is slightly decreased by the presence of FKBP51. Reported are average values of triplicate experiments ± SD. b The dynamic complex formed by Hsp90/Tau dissociates on the SEC column. c The presence of FKBP51 stabilizes the ternary complex. Fractions corresponding to the ternary complex (represented by asterisks) were submitted to SDS-PAGE showing the presence of the three proteins in the Hsp90/FKBP51/Tau and Hsp90/FKBP51/K32 ternary complexes. Note that FKBP51 and 441-residue Tau co-migrate in the gel resulting in overlapping bands on SDS-PAGE. Both full-length Tau and the K32 fragment¹⁶ were used, to illustrate the reproducibility of the results. MALS fittings for molecular weight estimation are indicated. Note that Tau retention times are comparable to those of much larger proteins, due to its disordered structure. The uncropped gel is shown in Supplementary Figure 8. d NMR interaction profiles of 441-residue Tau with Hsp90 (orange; Tau/Hsp90 molar ratio of 1:2), FKBP51 (bottom; Tau/FKBP51 molar ratio of 1:5) and the Hsp90/FKBP51 complex (top). I₀ and I are intensities of H–N cross-peaks of Tau in the absence and presence of the binding partner, respectively. For comparison, the binding pattern to Hsp90 is represented in the Tau/FKBP51 and Tau/Hsp90/FKBP51 plots by orange lines. The region covered by K32 fragment is shown on top. e Hydrophobic residues (green) in the N/M domains of Hsp90. f PREs induced in Hsp90 domains (red) by binding of Tau tagged with MTSL at position 256 within the binary Hsp90/Tau complex. g Phosphorylation of Tau impedes binding to Hsp90 and FKBP51. Cartoon representation indicating KXGS phosphorylation sites in Tau (green⁴¹), as well as sites for Ser-Thr pseudo-phosphorylation detected by AT8, AT100, and PHF1 antibodies (purple⁴²). h NMR binding plots of Ser-Thr pseudo-phosphorylated (purple) and MARK2-phosphorylated (green) Tau to Hsp90 (left) and FKBP51 (right). For comparison, the binding profiles of wild-type Tau (dotted lines) are shown