Atomistic and Thermodynamic Analysis of N6-Methyladenosine (m⁶A) Recognition by the Reader Domain of YTHDC1

N6-Methyladenosine (m6A) is the most frequent modification in eukaryotic messenger RNA (mRNA) and its cellular processing functions are regulated by reader proteins YTHDCs YTHDFs. However, mechanism of m6A recognition still elusive. Here, we investigate this process combining atomistic simulations, site-directed mutagenesis, biophysical experiments using YTHDC1 as a model. We find that N6 methyl group contributes to binding through specific interactions with an aromatic cage (formed Trp377 Trp428) also favoring association-prone conformation m6A-containing solution. The site dynamically equilibrates between multiple metastable conformations four residues being involved regulation (Trp428, Met438, Ser378, Thr379). Trp428 switches two conformational states build dismantle cage. Interestingly, mutating Met438 Ser378 alanine does not alter protein but significantly redistributes enthalpy entropy terms, i.e., enthalpy–entropy compensation. Such compensation reasoned different entropy–enthalpy transduction associated both changes wild-type mutant redistribution water molecules. In contrast, point Thr379Val thermal stability capability natural ligand. Additionally, thermodynamic analysis free energy calculations shed light on role structural molecule synergistically binds acts hub hydrogen-bond network. Taken together, experimental data simulation results may accelerate discovery chemical probes, m6A-editing tools, drug candidates against proteins.