Transient interdomain interactions shape the conformational ensemble governing RNA recognition by the tandem RRMs of Sex-lethal

RNA recognition motif (RRM) proteins often have multiple RNA-binding domains connected by flexible linkers, but how transient interdomain interactions affect RNA recognition is unclear. Here, the authors used solution NMR spectroscopy, rational protein engineering, restrained docking, and RNA-binding assays to investigate the tandem RRMs of the Drosophila melanogaster splicing regulator Sex-lethal (Sxl). Progressive extension of the native interdomain linker led to a gradual decrease in rotational coupling between the two RRMs and continuous chemical shift changes, indicating that the RNA-free protein samples a dynamic conformational ensemble rather than behaving as independently tumbling domains.

NMR-guided docking identified a compact arrangement compatible with the data and suggested a transient interface partially overlapping the RNA-binding surfaces. Surprisingly, a mutant designed to weaken this interface had the opposite effect: instead of increasing interdomain mobility, it enhanced rotational coupling while remaining natively folded, indicating redistribution of the conformational ensemble rather than disruption of domain architecture. Both linker extension and the mutant reduced RNA-binding affinity, and the mutant also diminished sequence discrimination. These results demonstrate that RNA recognition by Sxl is governed by a finely balanced conformational ensemble, and perturbing this equilibrium in either direction compromises high-affinity, sequence-selective RNA binding.