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. 2019 May 22;10(26):6503-6507.
doi: 10.1039/c9sc00531e. eCollection 2019 Jul 14.

Residue-specific identification of phase separation hot spots of Alzheimer's-related protein tau

Susmitha Ambadipudi  1 , Jithender G Reddy  2   3 , Jacek Biernat  4 , Eckhard Mandelkow  4   5 , Markus Zweckstetter  1   2
Affiliations

Residue-specific identification of phase separation hot spots of Alzheimer's-related protein tau

Susmitha Ambadipudi et al. Chem Sci. .

Abstract

Liquid-liquid phase separation (LLPS) of proteins enables the formation of non-membrane-bound organelles in cells and is associated with cancer and neurodegeneration. Little is known however about the structure and dynamics of proteins in LLPS conditions, because of the polymorphic nature of liquid-like protein droplets. Using carbon-detected NMR experiments we here show that the conversion of the aggregation-prone repeat region of the Alzheimer's-related protein tau from the dispersed monomeric state to phase-separated liquid-like droplets involves tau's aggregation-prone hexapeptides and regulatory KXGS motifs. Droplet dissolution in presence of 1,6-hexanediol revealed that chemical shift perturbations in the hexapeptide motifs are temperature driven, while those in KXGS motifs report on phase separation. Residue-specific secondary structure analysis further indicated that tau's repeat region exists in extended conformation in the dispersed state and attains transient β-hairpin propensity upon LLPS. Taken together our work shows that NMR spectroscopy can provide high-resolution insights into LLPS-induced changes in intrinsically disordered proteins.

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Figures

Fig. 1
Fig. 1. NMR spectroscopy of liquid–liquid phase separation of the repeat domain of tau. (a and b) Superposition of 2D 1H/15N-HSQC (a) and 2D CON (b) spectra of K18 in the monomeric dispersed state (5 °C, black) and the droplet phase (37 °C, red). To avoid contributions of solvent exchange to NMR signal broadening in 1H/15N correlation spectra, D2O was placed into a separate capillary tube (insert in a). (c) Superposition of CON spectra of K18 recorded at 37 °C in the absence (–hex, red) and presence (+hex, blue) of 3% 1,6-hexanediol, which rapidly dissolved K18 droplets (e). (d) DIC microscopy demonstrates K18 droplet formation at 37 °C. (e) Time-dependent dissolution of K18 droplets in presence of 3% 1,6-hexanediol observed by DIC microscopy.
Fig. 2
Fig. 2. Residue-specific analysis of LLPS-induced changes in chemical environment and local structure in the repeat-region of tau. (a) Domain representation of K18. Pseudo-repeats are shown as R1–R4, hexapeptides are marked in grey, KXGS motifs in red. (b) Chemical shift perturbation (CSP) of CO (red) and N (grey) from the dispersed monomeric (5 °C) to the droplet state (37 °C). (c) CSP of CO and N from the dispersed monomeric state (5 °C) to the dispersed monomeric state at 37 °C (i.e. in the presence of 1,6-hexanediol), revealing temperature-induced changes in N chemical shifts. (d) Averaged CSP (ΔCSP) of CO and N in K18 between the droplets state (without 1,6-hexanediol at 37 °C) and after droplet dissolution (with 1,6-hexanediol at 37 °C). The four KXGS motifs (red bars), as well as the proline-rich region and the PGGG motif (asterisk) in R1 display pronounced ΔCSP. (e) Secondary structure propensity of K18 in the monomeric dispersed state at 5 °C (black) and the LLPS-phase at 37 °C (yellow). At 37 °C, secondary chemical shifts suggest the presence of β-hairpin-like conformations in repeats R2, R3 and R4 (illustrated by a cartoon diagram on top).
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