Dynamic RRM Motions Encode Divergent Oligomerization Paths of hnRNP A2/B1 and A1 During Nucleic Acid Recognition

HnRNP A2/B1 and A1 are highly homologous RNA-binding proteins that exhibit striking functional divergence despite a conserved domain architecture. The mechanistic basis for this functional specialization remains unclear. Here, using isolated tandem RNA recognition motif (RRM) domains in integrated biophysical assays and molecular dynamics (MD) simulations, we show that their distinct oligomerization behaviors correlate with divergent inter-domain conformational landscapes. We find that A2/B1 adopts a rigid architecture, stabilized by a persistent network of electrostatic and hydrophobic interactions at the domain interface. This rigidity facilitates a U-shaped substrate topology, enabling homodimerization on short nucleic acids (12-nt). In contrast, A1 exhibits pronounced inter-domain flexibility and conformational heterogeneity. This structural plasticity disfavors stable dimerization, but enables A1 to engage extended substrates (≥17-nt) in a tandem manner. Mutational analysis confirms that disruption of the key interfacial interactions in A2/B1 induces A1-like dynamics and impairs dimerization. Together, these findings suggest that inter-domain dynamics serve as a key determinant of oligomerization pathways in multi-RRM proteins, providing a mechanistic framework for understanding how conserved RNA-binding modules can be functionally tuned.