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

Abstract

N6-Methyladenosine (m 6 A) is the most frequent modification in eukaryotic messenger RNA (mRNA) and its cellular processing and functions are regulated by the reader proteins YTHDCs and YTHDFs. However, the mechanism of m 6 A recognition by the reader proteins is still elusive. Here, we investigate this recognition process by combining atomistic simulations, sitedirected mutagenesis, and biophysical experiments using YTHDC1 as a model. We find that the N6 methyl group of m 6 A contributes to the binding through its specific interactions with an aromatic cage (formed by Trp377 and Trp428) and also by favoring the association-prone conformation of m 6 A-containing RNA in solution. The m 6 A binding site dynamically equilibrates between multiple metastable conformations with four residues being involved in the regulation of m 6 A binding (Trp428, Met438, Ser378, and Thr379). Trp428 switches between two conformational states to build and dismantle the aromatic cage. Interestingly, mutating Met438 and Ser378 to alanine does not alter m 6 A binding to the protein but significantly redistributes the binding enthalpy and entropy terms, i.e., enthalpy−entropy compensation. Such compensation is reasoned by different entropy− enthalpy transduction associated with both conformational changes of the wild-type and mutant proteins and the redistribution of water molecules. In contrast, the point mutant Thr379Val significantly changes the thermal stability and binding capability of YTHDC1 to its natural ligand. Additionally, thermodynamic analysis and free energy calculations shed light on the role of a structural water molecule that synergistically binds to YTHDC1 with m 6 A and acts as the hub of a hydrogen-bond network. Taken together, the experimental data and simulation results may accelerate the discovery of chemical probes, m 6 A-editing tools, and drug candidates against reader proteins.

Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1

“…As expected the binding enthalpy with the hairpin RNA is slightly reduced compared to the single-stranded m 6 A RNA due to the local unfolding of the closing base pair. Interestingly, recognition of m 6 A in the context of both the single-stranded or the hairpin RNA ligand by the YTH domain shows slightly unfavorable binding entropy (consistent with previous reports), although the release of solvent molecules from the pocket of the protein that accommodates the N 6 methyl is expected to show favorable entropic contributions for binding ( 60 , 61 ).…”

Recognition of N6-Methyladenosine by the YTHDC1 YTH Domain Studied by Molecular Dynamics and NMR Spectroscopy: The Role of Hydration

“…Methylamine indicates that twenty methylamines were added into the simulation water box before start. b NOEfix function was applied to all (312) or selected (18) experimental NOE distances showing ensemble-averaged distance violations in the 2mtv_m 6 A 3 simulations. c Modified vdW parameters of the phosphates 35 with associated modified RNA backbone dihedral parameters 36 and OPC water model 34 were used.…”

“…Indeed, a recent computational study of this system (using a different force field) utilized thermodynamic integration (TI) and indicated 1.7 kcal/mol penalty for binding of the unmethylated substrate. 18 However, the calculations were performed for a model system with an isolated m 6 A nucleoside rather than a full oligonucleotide and the solute atoms were immobilized by positional restraints. 18 Furthermore, our MD simulations indicated that a single water molecule often invades the binding pocket in presence of standard adenosine (see above and Figure 3) which somewhat complicates the sampling of m 6 A 3 A 3 TI calculations; see Supporting Information for further details.…”

“…In the past, the MD simulations have been successfully applied many times in studies of protein/RNA complexes, 16 including the YTH domain. 14,[17][18] By providing essentially infinite temporal and spatial resolution, the MD can extend upon the information from ensemble-averaged experimental data. It is particularly well-suited for studies of hydration and its structural role in biomolecular complexes.…”

Mutant NPM1-Regulated FTO-Mediated m6A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

“…Meanwhile, m 6 A modifications can be removed by a-ketoglutarate-dependent and Fe(II)-dependent demethylases, specifically fat mass and obesity-associated protein (FTO) (20) and alkB homolog 5 (ALKBH5) (21). Additionally, the functions of m 6 A modifications on RNA metabolism depend on specific recognition by m 6 A binding proteins (YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, and hnRNPC) (22)(23)(24). A rapidly accumulating wealth of studies are delineating the aberrant m 6 A abundance driven by the dysregulation of m 6 A modification enzymes in diverse cancers.…”