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The molecular mechanism of temperature-dependent phase separation of heat shock factor 1

Nature Chemical Biology volume 21, pages 831–842 (2025)Cite this article

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Abstract

Heat shock factor 1 (HSF1) is the critical orchestrator of cell responses to heat shock, and its dysfunction is linked to various diseases. HSF1 undergoes phase separation upon heat shock, and its activity is regulated by post-translational modifications (PTMs). The molecular details underlying HSF1 phase separation, temperature sensing and PTM regulation remain poorly understood. Here, we discovered that HSF1 exhibits temperature-dependent phase separation with a lower critical solution temperature behavior, providing a new conceptual mechanism accounting for HSF1 activation. We revealed the residue-level molecular details of the interactions driving the phase separation of wild-type HSF1 and its distinct PTM patterns at various temperatures. The mapped interfaces were validated experimentally and accounted for the reported HSF1 functions. Importantly, the molecular grammar of temperature-dependent HSF1 phase separation is species specific and physiologically relevant. These findings delineate a chemical code that integrates accurate phase separation with physiological body temperature control in animals.

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Fig. 1: The RD region of HSF1 drives temperature-dependent phase separation.
Fig. 2: HSF1 undergoes an LCST-type phase transition attributed to solvent entropy changes.
Fig. 3: The intermolecular interactions among RD domains in solution are modulated by temperature and variants, as revealed by NMR.
Fig. 4: NMR data-driven CG simulations reproduced HSF1 temperature-dependent phase separation.
Fig. 5: Regions of intermolecular interaction mediate the phase separation of HSF1.
Fig. 6: Regions with temperature-dependent intermolecular interactions determine the phase transition temperature of HSF1.

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Data availability

The authors declare that all the data associated with the figures are provided as Supplementary Information. Source data are provided with this paper. All the experimental and simulation raw data are available at https://doi.org/10.5281/zenodo.13895417 (ref. 79). Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.

Code availability

The code can be downloaded from https://github.com/carolinge/HSF_MD.

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Acknowledgements

We thank the staff and G. Liu from the BL19U2 beamline(https://cstr.cn/31129.02.NFPS.BL19U2) of the National Facility for Protein Science in Shanghai (https://cstr.cn/31129.02.NFPS) at the Shanghai Synchrotron Radiation Facility for providing technical support and assistance in SAXS data collection and analysis. This work was supported by grants from the National Natural Science Foundation of China (32090040 to S.X., Z.H. and Y.X.; 31971128 to S.X.; 92254302 to Y.X.) and the National Key R&D Program (2019YFA0508403 to S.X.; 2022YFA1303100 to Z.H. and Y.X.) and the Strategic Priority Research Program of the Chinese Academy of Sciences Grant no. XDB 37040202 to S.X. and CAS Project for Young Scientists in Basic Research, Grant no. YSBR-068 to S.X.

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Author notes

  1. These authors contributed equally: Qiunan Ren, Linge Li.

Authors and Affiliations

  1. MOE Key Laboratory for Cellular Dynamics, School of Life Sciences, University of Science and Technology of China, Hefei, China

    Qiunan Ren, Lei Liu, Juan Li, Xuebiao Yao & ShengQi Xiang

  2. Hefei National Research Center for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China

    Linge Li, Chaowei Shi, Xuebiao Yao & Zhonghuai Hou

  3. Department of Chemical Physics, University of Science and Technology of China, Hefei, China

    Linge Li & Zhonghuai Hou

  4. National Biomedical Imaging Center, College of Future Technology, Peking University, Beijing, China

    Yujie Sun

  5. State Key Laboratory of Membrane Biology & Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China

    Yujie Sun

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Contributions

Q.R., L. Li, X.Y., Z.H. and S.X. conceived and designed the experiments. Q.R. performed most of the biochemical and NMR experiments. L. Li performed the coarse-grained simulation. L. Liu assisted with purification of the IDR of HSF1. J.L. and C.S. helped with the NMR experiments. Y.S. provided the original plasmids of HSF1. Q.R., L. Li, X.Y., Z.H. and S.X. wrote the manuscript.

Corresponding authors

Correspondence to Xuebiao Yao, Zhonghuai Hou or ShengQi Xiang.

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Extended data

Extended Data Fig. 1 Temperature effects on phase transition and aggregation of HSF1.

a. The correlation between species body temperature and the middle point temperature of the phase transition curve. b. The box-plots of droplet sizes in Fig. 1e. n = 79 for WT-25 °C, n = 100 for WT-37 °C, n = 71 for M1-5 °C, n = 94 for M1-25 °C and n = 85 for M1-37 °C across 3 independent experiments. ND, not determined. c. The peak with a retention time of 10 ml was the LZ1-3 oligomers by SEC-MALS. The UV absorbance values were in red, and the light scattering values were in gray. The molar mass values (g/mol) were in blue. d. DSS cross-linked lz1-3 at different temperatures were detected by SDS‒PAGE, which is representative of 3 independent experiments. e. The box-plots of droplet sizes in Fig. 1f. n = 86 for WT-25 °C, n = 190 for WT-37 °C, n = 153 for M1-5 °C, n = 153 for M1-25 °C and n = 151 for M1-37 °C across 3 independent experiments. ND, not determined. In the box plots, data are presented as mean values, box plots: 25th to 75th percentiles, median line, 1.5× interquartile as whiskers and points.

Source data

Extended Data Fig. 2 The backbone assignments of RD(WT), RD(M1) and RD(M2).

The backbone assignments were plotted on the 1H-15N HSQC, which were recorded at 5 °C.

Source data

Extended Data Fig. 3 Comparative NMR analysis of wild-type and mutant RD domains.

a. The secondary chemical shifts of WT RD, M1 and M2. b. Secondary structure populations of WT RD, M1 and M2 obtained from the delta2D method using experimentally assigned backbone chemical shifts. c. The hetNOE values of the backbone amide groups of WT RD, M1 and M2. The light orange columns represent the WT, while the red and blue lines depict M1 and M2, respectively. d. T1 and e. T2 of WT RD, M1 and M2. WT, gray line. M1, red line. M2, blue line. NMR data were collected at 5 °C.

Source data

Extended Data Fig. 4 Temperature-dependent NMR chemical shifts and SAXS-derived Rg values of RD protein variants: WT, M1, and M2.

a-c.The temperature coefficient of the 1HN chemical shift values of the backbone amide groups of WT(a), M1(b), and M2(c) at different temperature ranges. d. Rg values of WT RD, M1, and M2 mutants were measured by SAXS at 5 °C and 37 °C. At each temperature for each sample, a set of 20 sample curves and their corresponding 20 buffer curves were collected. The software processed these data by averaging the 20 sample curves and then subtracting the average background curve to eliminate any baseline effects. Subsequently, the mean radii of gyration (Rg), along with their associated errors, were calculated through multiple Guinier fits. Consequently, the figure presents the result of the collective average curve rather than plotting individual data points.

Source data

Extended Data Fig. 5 Analysis of Mean-Field Model parameters and solution viscosity of different conditions.

a. Fitting parameters for the mean-field model. b, c. The diffusion coefficient of 1,4-Dioxane in 0.1 mM and 1 mM RD at 5 °C and 30 °C using DOSY.

Source data

Extended Data Fig. 6 NMR intensities ratios reveal the intermolecular interactions between RD domains.

The NMR signal intensity ratio of every residue of the WT RD(a), M1(b) and M2(c) when it was alone or in the presence of 10-fold excess unlabeled counterpart at 15 °C and 25 °C. All error bars were calculated according to the signal-to-noise ratio of the spectra, as explained in the methods section. d-e. The NMR spectra slices along 1HN-dimension of M212 and A329 of WT RD from 5 °C to 30 °C.

Source data

Extended Data Fig. 7 The linewidth distribution of peaks in 15N-RD and 15N + 14N-RD samples at 5 °C (a) and 30 °C (b).

In both correlation plots, the dots on the upper right are linewidth values in the 1H dimension, and the dots on the lower left are linewidth values in the 15N dimension. The linear regression analysis results were shown on each plot.

Source data

Extended Data Fig. 8 Sequence analysis with localCIDER and phase separation simulation of RD protein variants with different force fields.

a. Sequence analysis results of some commonly applied sequence parameters, calculated with localCIDER b. The simulation using existing force fields failed to reproduce the phase separation. The experimental data of WT and M1 are shown in gray and red dots. The current NMR-driven CG simulation predicted values at the 4 temperatures (5 °C, 15 °C, 25 °C and 30 °C) are shown in gray and red crosses.

Source data

Extended Data Fig. 9 Force field optimization and Coarse-Grained simulation of protein phase separation.

a. Normalized difference between simulated and experimental HSQC values \(\langle \tfrac{|NM{R}_{\exp }^{i}-NM{R}_{{\rm{sim}}}^{i}|}{NM{R}_{\exp }^{i}}\rangle\) as a function of the number of iterations for force field optimization. b. Comparison between experimental HSQC and simulated HSQC after force field optimization. c. Snapshots of the CG simulation, which reproduced the experimental phase separation observation of LZ1-3-RD at 15 °C and 25 °C. d. Distributions of the reduced density ρ of WT, M1, and M2 in the CG simulation at 15 °C and 25 °C. e. Changes in the protein amounts in the largest condensate ρ of WT, M1, and M2 during CG simulation at 15 °C and 25 °C.

Source data

Extended Data Fig. 10 Schematic representation of peptide mutants in biochemical assays and interspecies sequence variation in HSF1 LZ1-3-RD-LZ4 domains.

Sequence schemes of RD(WT) aa224-248-S(a) and RD(M1) aa320-343-S(b) mutants. The positively charged residues were labeled in red and the negatively charged residues were labeled in blue, while the hydrophilic mutations are in black. c. The sequence alignment of the LZ1-3-RD-LZ4 regions from HSF1 of humans, mice, chicken, and zebrafish showed the main species-specific differences between residue 320 and 360, matching with the regions with a temperature-dependent interaction pattern in Fig. 5a.

Supplementary information

Source data

Source Data Fig. 1

Statistical source data for the turbidity curves of LZ1−3-RD-LZ4 from different species.

Source Data Fig. 2

Source data for phase diagrams and water and entropy statistics for a,c,d. Statistical source data for droplet sizes of RD(M1)-L.

Source Data Fig. 3

Source data for the NMR signal intensity ratio of every residue at 5 °C and 30 °C.

Source Data Fig. 4

Statistical source data for density, cluster growth and Rg distributions for panels c−e.

Source Data Fig. 5

Statistical source data for droplet sizes.

Source Data Fig. 6

Statistical source data for the turbidity−temperature curves.

Source Data Extended Data Fig. 1

Statistical source data for droplet sizes and SEC−MALS of LZ1−3.

Source Data Extended Data Fig. 2

Source data for the backbone assignments of WT, M1 and M2 RD.

Source Data Extended Data Fig. 3

Source data for the secondary chemical shifts, the secondary structure populations, the hetNOE values, T1 and T2 of WT, M1 and M2 RD.

Source Data Extended Data Fig. 4

Source data for temperature-dependent NMR chemical shifts and SAXS-derived Rg values of WT, M1 and M2 RD.

Source Data Extended Data Fig. 5

Source data for the diffusion coefficient of 1,4-dioxane in 0.1 mM and 1 mM RD at 5 °C and 30 °C.

Source Data Extended Data Fig. 6

Source data for the NMR signal intensity ratio of every residue at 15 °C and 25 °C.

Source Data Extended Data Fig. 7

Statistical source data for the linewidth distribution of peaks in 15N-labeled RD and 15N + 14N-labeled RD samples at 5 °C and 30 °C.

Source Data Extended Data Fig. 8

Source data for phase diagrams of other force fields.

Source Data Extended Data Fig. 9

Statistical source data for simulated HSQC data and density, cluster growth and distributions for b,d,e.

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Ren, Q., Li, L., Liu, L. et al. The molecular mechanism of temperature-dependent phase separation of heat shock factor 1. Nat Chem Biol 21, 831–842 (2025). https://doi.org/10.1038/s41589-024-01806-y

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