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

Heat shock factor 1 (HSF1) is the critical orchestrator for a cell responding to heat shock, and its dysfunction is linked to cancers and neurodegenerative diseases. HSF1 undergoes phase separation upon heat shock, and its activity is regulated by various post-translational modifications (PTMs). Despite its importance, the molecular details underlying HSF1 phase separation, temperature sensing, and post-translational modifications (PTMs) regulation remain poorly understood. Here, we discovered that HSF1 exhibits temperature-dependent phase separation with a lower critical solution temperature (LCST) behavior due to entropy contribution from solvent molecules, providing a new conceptual mechanism accounting for HSF1 activation. We employed a synergistic approach combining coarse-grain simulation and nuclear magnetic resonance spectroscopy to reveal 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 identified interaction sites were further validated with biochemistry assays and mapped interface accounts for HSF1 functions reported. Importantly, the amino acid substitution experiment reveals the molecular grammar for temperature-dependent HSF1 phase separation is species-specific and physiologically relevant. These findings delineate chemical code that integrates protein PTM patterns with accurate phase separation for body physiological temperature control in animals.