SAXS reveals the molecular basis underlying pH-driven G3BP1 conformational dynamics: implications for stress granule formation

Stress granules (SGs) are cytoplasmic condensates of proteins and RNAs that form in response to cellular stress, playing a critical role in protecting cells during adverse conditions. G3BP proteins act as a central node and molecular switch in SG assembly. Growing evidence indicates that conformational changes in G3BP1 mediate its phase separation competence. However, the mechanistic basis underlying how G3BP conformational dynamics contribute to SG formation has remained elusive. Here, we demonstrate that the G3BP1 homodimer adopts both distinct expanded and compact conformations in solution, with transitions finely modulated by physiological changes in ionic strength and pH. Notably, the G3BP1(dRGG) deletion mutant, which is SG-incompetent, abolishes environmentally induced conformational transitions, underscoring the pivotal regulatory role of the RGG domain in G3BP1 dynamics. Additionally, G3BP1 can undergo RNA-independent phase separation at pH 6. Our findings establish a structural framework that connects the conformational plasticity of G3BP1 to its capacity for condensate assembly. We propose that the interplay between intrinsically disordered regions (IDRs), regulated by pH-driven conformational dynamics of G3BP1, finely tunes the threshold for initial granule assembly. These insights significantly enhance our understanding of SG dynamics and provide a mechanistic foundation for investigating how environmental stress conditions influence SG formation and function.