  N6-methyladenosine (m6A) is a prevalent RNA post-transcriptional modification
that plays crucial roles in RNA stability, structural dynamics, and
interactions with proteins. The YT521-B (YTH) family of proteins, which are
notable m6A readers, function through their highly conserved YTH domain. Recent
structural investigations and molecular dynamics (MD) simulations have shed
light on the recognition mechanism of m6A by the YTHDC1 protein. Despite
advancements, using MD to predict the stabilization induced by m6A on the free
energy of binding between RNA and YTH proteins remains challenging, due to
inaccuracy of the employed force field and limited sampling. For instance,
simulations often fail to sufficiently capture the hydration dynamics of the
binding pocket. This study addresses these challenges through an innovative
methodology that integrates metadynamics, alchemical simulations, and
force-field refinement. Importantly, our research identifies hydration of the
binding pocket as giving only a minor contribution to the binding free energy
and emphasizes the critical importance of precisely tuning force-field
parameters to experimental data. By employing a fitting strategy built on
alchemical calculations, we refine the m6A partial charges parameters, thereby
enabling the simultaneous reproduction of N6 methylation on both the protein
binding free energy and the thermodynamic stability of nine RNA duplexes. Our
findings underscore the sensitivity of binding free energies to partial
charges, highlighting the necessity for thorough parameterization and
validation against experimental observations across a range of structural
contexts.
