RNA recognition motif (RRM) proteins represent an abundant class of proteins playing key roles in RNA biology. We present a joint atomistic molecular dynamics (MD) and experimental study of two RRM-containing proteins bound with their single-stranded target RNAs, namely the Fox-1 and SRSF1 complexes. The simulations are used in conjunction with NMR spectroscopy to interpret and expand the available structural data. We accumulate more than 50 μs of simulations and show that the MD method is robust enough to reliably describe the structural dynamics of the RRM-RNA complexes. The simulations predict unanticipated specific participation of Arg142 at the protein-RNA interface of the SRFS1 complex, which is subsequently confirmed by NMR and ITC measurements. Several segments of the protein-RNA interface may involve competition between dynamical local substates rather than firmly formed interactions, which is indirectly consistent with the primary NMR data. We demonstrate that the simulations can be used to interpret the NMR atomistic models and can provide qualified predictions. Finally, we propose a protocol for 'MD-adapted structure ensemble' as a way to integrate the simulation predictions and expand upon the deposited NMR structures. Unbiased μs-scale atomistic MD could become a technique routinely complementing the NMR measurements of protein-RNA complexes.

Institute of Biophysics Academy of Sciences of the Czech Republic Kralovopolska 135 612 65 Brno Czech Republic

Institute of Biophysics Academy of Sciences of the Czech Republic Kralovopolska 135 612 65 Brno Czech Republic CEITEC Central European Institute of Technology Masaryk University Campus Bohunice Kamenice 5 625 00 Brno Czech Republic

Institute of Molecular Biology and Biophysics Department of Biology ETH Zurich CH 8093 Zurich Switzerland

Institute of Molecular Biology and Biophysics Department of Biology ETH Zurich CH 8093 Zurich Switzerland Global Discovery Chemistry Novartis Institute for BioMedical Research Basel CH 4002 Switzerland

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An intricate balance of hydrogen bonding, ion atmosphere and dynamics facilitates a seamless uracil to cytosine substitution in the U-turn of the neomycin-sensing riboswitch

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Structural study of the Fox-1 RRM protein hydration reveals a role for key water molecules in RRM-RNA recognition