Conformational Heterogeneity of RNA Stem-Loop Hairpins Bound to FUS RNA Recognition Motif with Disordered RGG Tail Revealed by Unbiased Molecular Dynamics Simulations.

RNA-protein complexes use diverse binding strategies, ranging from structurally well-defined interfaces to completely disordered regions. Experimental characterization of flexible segments is challenging and can be aided by atomistic molecular dynamics (MD) simulations. Here we used extended set of microsecond-scale MD trajectories (400 s in total) to study two FUS-RNA constructs previously characterized by NMR spectroscopy. The FUS protein contains well-structured RNA Recognition Motif domain followed by presumably disordered RGG tail and bind RNA stem-loop hairpins. Our simulations provide several suggestions complementing the experiments but also reveal major methodological difficulties in studies of such complex RNA-protein interfaces. Despite efforts to stabilize the binding via system-specific force-field adjustments, we have observed progressive distortions of the RNA-protein interface inconsistent with experimental data, as in detail documented. We further propose that the dynamics is so rich that its converged description would not be achievable even upon stabilizing the system. Still, after careful analysis of the trajectories, we have made several suggestions regarding the binding. We identify substates in the RNA loops which can explain the NOE data. The RGG tail localized in the minor groove remains disordered, sampling countless transient interactions with the RNA. There are long-range couplings among the different elements contributing to the recognition, which can lead to allosteric communication throughout the system. Overall, the RNA-FUS systems form dynamical ensembles that cannot be fully represented by single static structures. Thus, albeit imperfect, MD simulations represent a viable tool to investigate them.