Conformational plasticity modulates sequence specificity in non-canonical tandem RRM-RNA binding

Abstract

The Dead End protein (DND1), a key regulator of germline cell fate, utilizes two RNA Recognition Motifs (RRM) in tandem to bind AU-rich RNA in a non-canonical 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 inter-domain 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 non-cognate RNA sequences. This substantiates our findings which showcase how structural dynamics impact RNA recognition, enabling structural adaptation for functional versatility in multi-domain proteins. Statement of SignificanceThe RNA recognition motif (RRM) is the most abundant RNA binding domain in proteins. Often, one protein has multiple RRMs to enhance RNA binding specificity. For example, Dead End, an essential protein in germ cells with context dependent roles in cancer has two RRMs connected by a short linker. It is not understood how the inter-domain dynamics between the two RRMs modulate the RNA binding specificity. We found that the tandem RRMs of Dead End display extensive conformational plasticity in molecular dynamics simulations with variable inter-domain orientations. Despite this plasticity, the core RNA binding specificity is kept as a result of the cooperative binding of the two RRMs. Our findings provide evidence for highly dynamic and adaptable RNA recognition by tandem RRMs.