Conformational plasticity and sequence specificity interplay in noncanonical tandem RRM-RNA binding.

Dead end protein 1 (DND1), a key regulator of germline cell fate, utilizes two RNA recognition motifs (RRMs) in tandem to bind AU-rich RNA in a noncanonical manner. Only one RRM uses the known RNA-binding interface, whereas the second motif has only minimal contacts with the RNA. To characterize the structural features and dynamics that contribute to RNA binding, we performed a series of atomistic molecular dynamics simulations and found that the complex is highly dynamic, deviating significantly from the experimental structure. We found that RNA binding restricts-without abolishing-the interdomain motions of the RRMs and that cooperative binding of both RRMs is required to reduce the flexibility of the bound RNA. Through detailed analysis of the protein-RNA interactions, we show that the RNA-binding interface in the tandem RRM-RNA complex remains stable despite extensive conformational plasticity. The RRMs cooperate to sustain a layered RNA-binding mechanism, with aromatic stacking of the central adenosine with RRM1 residues as foundation, arginine residues anchoring the RNA backbone, and residues in the inter-RRM linker forming hydrogen bonds with the RNA. Despite insufficient conformational space sampling to claim convergence, the protein-RNA interactions were consistent across multiple independent simulations with cognate and noncognate RNA sequences. This substantiates our findings, which showcase how structural dynamics impact RNA recognition, enabling structural adaptation for functional versatility in multidomain proteins.