The YTH domain of YTHDC1 belongs to a class of protein "readers", recognizing the N6-methyladenosine (m6A) chemical modification in mRNA. Static ensemble-averaged structures revealed details of N6-methyl recognition via a conserved aromatic cage. Here, we performed molecular dynamics (MD) simulations along with nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) to examine how dynamics and solvent interactions contribute to the m6A recognition and negative selectivity toward an unmethylated substrate. The structured water molecules surrounding the bound RNA and the methylated substrate's ability to exclude bulk water molecules contribute to the YTH domain's preference for m6A. Intrusions of bulk water deep into the binding pocket disrupt binding of unmethylated adenosine. The YTHDC1's preference for the 5'-Gm6A-3' motif is partially facilitated by a network of water-mediated interactions between the 2-amino group of the guanosine and residues in the m6A binding pocket. The 5'-Im6A-3' (where I is inosine) motif can be recognized too, but disruption of the water network lowers affinity. The D479A mutant also disrupts the water network and destabilizes m6A binding. Our interdisciplinary study of the YTHDC1 protein-RNA complex reveals an unusual physical mechanism by which solvent interactions contribute toward m6A recognition.

Department of Biology Institute of Biochemistry ETH Zürich 8093 Zürich Switzerland

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

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Regional Centre of Advanced Technologies and Materials Czech Advanced Technology and Research Institute Palacký University Olomouc Olomouc 783 71 Czech Republic

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