Structural and functional insights into a mesophilic cold shock protein CspA with enhanced precision

N CPMG and visible peak-position constraints approach to probe conformational exchange in CSPs. Our results show that, in addition to the conserved aromatic residues of RNP1 and RNP2 motifs that mediate π-stacking interactions with nucleic acids, an unexpectedly broad network of hydrophobic core and solvent-exposed polar residues undergoes conformational exchange. Notably, residues in the β3-β4 and β4-β5 loops display complex dynamics not fully captured by model-free analysis formalism, highlighting their role in binding site flexibility. Complementary AF3/YASARA modeling of the CspA bound to heptathymidine (dT7) further supported that aromatic and polar residues form π-stacking and ionic interactions with ssDNA bases, corroborating the functional relevance of these dynamic regions. Therefore, our findings demonstrate that CspA relies on a dynamic network extending from conserved motifs through the hydrophobic core and flexible loops, conferring the structural adaptability required for efficient nucleic acid recognition and chaperone activity.