Susmitha
Ambadipudi
a,
Jithender G.
Reddy
bc,
Jacek
Biernat
d,
Eckhard
Mandelkow
de and
Markus
Zweckstetter
*ab
aDeutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Von-Siebold-Str. 3a, 37075 Göttingen, Germany. E-mail: mazw@nmr.mpibpc.mpg.de
bMax-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany
cNMR & Structural Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
dDeutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany
eCAESAR Research Center, Bonn, MPI for Metabolism Research, Hamburg Outstation, 22607 Hamburg, Germany
First published on 22nd May 2019
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.
Pathogenic aggregation of the microtubule-associated protein tau is the hallmark of Alzheimer's disease and tauopathies.15,16 The 441-residue tau is the longest isoform in the adult human brain and comprises two N-terminal inserts, the proline-rich region and four pseudo-repeats in the C-terminal half.17–20 The four pseudo-repeats are important for microtubule-binding and aggregation.21 A truncated tau version that contains only the four pseudo-repeats (termed K18) has higher aggregation propensity than the full-length protein.22 We and others previously showed that both tau and K18 undergo LLPS in a pH- and temperature-dependent manner, droplet formation is modulated by phosphorylation at KXGS motifs and LLPS promotes tau fibrillization and microtubule polymerization.10–12,23
Here, we used 13C-detected NMR experiments to identify the key residues involved in phase separation of the four-repeat region of tau at high resolution. We show that the local chemical environment at tau's aggregation-prone hexapeptides and the regulatory KXGS motifs is altered upon phase separation and droplet formation. Residue stretches spanning these motifs gain transient β-hairpin propensity, while the protein retains the overall disordered nature upon LLPS.
The NMR spectra displayed temperature-dependent chemical shift changes with signal broadening varying across residues. Several of the 1H/15N signals became undetectable above 25 °C (Fig. S1a and b†). The experiments showed that LLPS of K18 induces – independent of rapid solvent exchange – strong NMR signal broadening in 1H/15N correlation spectra. Because little signal broadening was observed in 1H/13C HSQC spectra of phase-separated K18 recorded under identical solution conditions,10 inhomogeneity in magnetic susceptibility anisotropy can be excluded as a major source of broadening. Instead, chemical exchange between different conformations (potentially involving transient hydrogen bonds) might cause line broadening of 1H/15N cross peaks. Consistent with the LLPS-specific nature of NMR signal broadening, other intrinsically disordered proteins showed severe broadening in 1H/15N correlation spectra upon LLPS.7,24
In order to improve spectral resolution and minimize LLPS-induced NMR signal broadening in K18, we recorded 2D 13C-detected NMR experiments.2513CO/15N correlation spectra (CON) are particularly powerful, because the chemical shift dispersion of CO is larger when compared to HN and therefore CON spectra offer better spectral resolution (Fig. 1b and S1c†).26,27 In addition, CON cross-peaks are less affected by solvent exchange at high pH and temperature.25 CON spectra of K18 were recorded in both the monomeric dispersed (5 °C) and phase-separated state (37 °C), keeping sample conditions identical to those used for 1H/15N correlation spectra. Both CON spectra were well-resolved, showed little signal broadening and displayed chemical shift perturbations between 5 °C and 37 °C (Fig. 1b, S1c and d†).
We next asked if the chemical shift changes inherent to temperature changes can be distinguished from those due to LLPS. To this end, we added 1,6-hexanediol, an aliphatic alcohol, to K18 droplets preformed at 37 °C. 1,6-hexanediol dissolves liquid-like droplets in vitro and in cells through disruption of weak interactions between the constituent protein molecules.28 DIC microscopy demonstrated that addition of 3% 1,6-hexanediol dissolved K18 droplets within ∼2 minutes (Fig. 1e). Superposition of CON spectra in the phase-separated (37 °C, without 1,6-hexanediol) and droplet-dissolved states (37 °C, with 1,6-hexanediol) revealed small chemical shift changes (Fig. 1c), suggesting that the two states differ locally in structure and/or dynamics.
The high quality of the 13C-detected NMR experiments at 37 °C suggested that it might be possible to follow the chemical shift perturbations for individual residues, and thus gain residue-specific insights into K18 phase separation. To this end, we assigned the backbone resonances of K18 at 5 °C and pH 8.8, with the help of HNCO,29 HN(C)N,30 HNCACB and HN(CO)CACB31 experiments (Table S1†). The observed chemical shifts were used to assign the peaks (including proline residues) in the CON spectrum at 5 °C. We then recorded a series of CON spectra for increasing temperatures from 5 to 37 °C. High spectral quality was retained for all temperatures (Fig. S1c and d†), in contrast to 1H/15N correlation spectra, where broadening of NMR signals was severe in conditions of LLPS (37 °C) (Fig. S1a and b†). Many of the NMR signals that were broadened beyond detection in the 1H/15N spectra (Fig. S1b†) were from glycine and serine residues (Fig. S1d†). Subsequently, CON spectra at increasing temperatures were sequence-specifically assigned (including prolines) based on the resonance assignment at 5 °C.
As the dispersed protein proceeded to form droplets when reaching 37 °C, chemical shift perturbation was observed for most K18 residues (Fig. 1b, S1c and d†). Analysis of the perturbations for individual nuclei furthermore showed that CO and N display a distinct sequence behavior. In case of CO, the perturbations are larger in proximity to the KXGS motifs, while for N the perturbation was most pronounced at the hexapeptides (Fig. 2a and b). In presence of 1,6-hexanediol, where the droplets were dissolved, the perturbation (relative to 5 °C) in the hexapeptides was retained, reflecting that these changes are primarily caused by the change in temperature from 5 to 37 °C (Fig. 2c). 1,6-hexanediol showed little or no effect on the chemical shifts of the dispersed protein at 5 °C (Fig. S3†). The combined chemical shift difference plot with and without 1,6-hexanediol (i.e. droplets dissolved vs. K18 droplets at 37 °C) displayed perturbation in the KXGS motifs of K18 (Fig. 2d and S4†). In addition, the proline-rich region and the PGGG motif in repeat R1 were affected by LLPS (Fig. 2d). Consistent with the importance of the KXGS motifs for tau-LLPS, phosphorylation of serine residues in the KXGS motifs by MARK2, a kinase that is involved in regulation of microtubule binding of tau,32 promotes self-coacervation of tau.10,12
We further asked if the changes in chemical shift environment concomitantly induced local structural changes upon LLPS of K18. Our previous study based on amide H and N chemical shift dispersion and far UV circular dichroism of K18 at 37 °C suggested that the protein remains largely disordered within the droplets, in agreement with the intrinsically disordered nature of tau.10 However, these approaches did not allow a residue-specific analysis of the structural content in the phase-separated state. We therefore recorded 2D 13C-detected CBCACO spectra25 of K18 for increasing temperatures from 5 °C (dispersed state) to 37 °C (droplet state) (Fig. S5†). Because CA, CB and CO chemical shifts are sensitive to phi/psi dihedral angles, secondary chemical shifts (Δδ), estimated as the difference between observed and random coil chemical shift values, report on protein secondary structure with positive (negative) deviation of ΔδCA and ΔδCO indicating helical (β-structure) propensity.
First, the CBCACO spectrum at 5 °C was assigned using the assignment obtained at 5 °C by 1H-detected experiments (see above). Next, this assignment was used to follow the temperature-dependent chemical shift changes of CA, CB and CO resonances from 5 °C to 37 °C. Subsequently, temperature-dependent Δδ values of CA, CB and CO were estimated using random coil chemical shifts at pH 8.8 and the given temperature.33 At 5 °C, the secondary structural propensity, calculated as ΔδCA − ΔδCB was negative for almost all residues (Fig. 2e), suggesting an extended K18 conformation. Upon phase separation at 37 °C, V275-H299, S305-H330 and G334-I360, which include the hexapeptides and KXGS motifs, displayed an alternating negative/positive ΔδCA − ΔδCB pattern, as expected for β-hairpin structure (Fig. 2e). A similar trend was observed for CO secondary chemical shifts at 37 °C, in the absence of 1,6-hexanediol (Fig. S6a†). In presence of 1,6-hexanediol where the droplets were dissolved, more negative CO secondary chemical shifts were observed, indicating a higher tendency for extended structure (Fig. S6b†). In the pseudo-repeats R2 and R3, ‘X’ of the KXGS motif corresponds to C291 and C322, respectively. The observed CB chemical shifts of C291 (28.47 ppm) and C322 (28.48 ppm) are characteristic for a reduced thiol group.34 Protein dimerization as a source for the observed changes in secondary structure propensity could therefore be excluded.
A defining property of intrinsically disordered proteins is that they can rapidly exchange between different conformations in solution.35 In this situation, the magnitude of Δδ will be reduced when compared to well-ordered proteins. In conditions of LLPS, the protein also can exchange between the droplet interior and the surrounding environment, potentially resulting in further chemical shift averaging (when the exchange is fast on the NMR time scale). The small changes in secondary structure propensity that were observed for K18 upon LLPS (Fig. 2e) are thus in agreement with the dynamic and polymorphic nature of phase separation of intrinsically disordered proteins. The propensity for β-hairpin formation in repeats 2, 3 and 4 upon LLPS suggests that LLPS favors amyloid promoting structure of tau. Conversion of liquid-like droplets into hydrogels might further stabilize the transient β-hairpin conformation and result in cross-β-structure in amyloid fibrils as previously observed for FUS protein.36
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc00531e |
This journal is © The Royal Society of Chemistry 2019 |