SAXS reveals the molecular basis underlying pH-driven G3BP1 conformational dynamics: implications for stress granule formation
G3BP1 is the central node and molecular switch in stress granule (SG) assembly. However, structural insights into full-length G3BP1 remain elusive owing to its extensive intrinsically disordered regions (IDRs). Using size-exclusion chromatography-coupled small-angle X-ray scattering (SEC-SAXS), we have characterized the solution architecture and conformational dynamics of full-length G3BP1. Under physiological conditions, G3BP1 adopts an elongated, head-to-head antiparallel homodimeric conformation, whereas acidification induces a pronounced conformational compaction. Subsequent biophysical studies reveal that this compact state enables robust RNA-mediated and, notably, homotypic phase separation in vitro. Deletion of the RGG region abolishes this acidity-induced compaction and markedly impairs phase separation, establishing a causal link between the RGG-dependent conformational switch and phase separation propensity. By moving beyond hypothetical models to experimental solution-state data, our work fills a longstanding void in the field and provides critical insights into the structural plasticity that underlies G3BP1 function, offering a missing structural link essential for deciphering the molecular mechanism of SG formation. We propose that stress-associated physicochemical changes, specifically localized acidification coupled with mRNA accumulation, trigger this reversible structural reconfiguration of G3BP1, thereby facilitating phase separation.