Recognition of N6-methyladenosine by the YTHDC1 YTH domain studied by molecular dynamics and NMR spectroscopy: The role of hydration

Background 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.

Methods We performed molecular dynamics (MD) simulations along with nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) measurements to examine how dynamics and solvent interactions contribute to the m⁶A recognition and negative selectivity towards unmethylated substrate.

Results An intricate network of water-mediated interactions surrounds bound m⁶A. The unmethylated adenosine allows disruptive intrusions of bulk water deep into the binding pocket, increasing selectivity for m⁶A. We furthermore show that 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 preceding guanosine and residues deep in the m⁶A binding pocket. The 5′-Im⁶A-3′ (where I is inosine) motif can be recognized as well at the cost of disrupting the water network and a small decrease in affinity. The YTHDC1 D479A mutant, which interrupts the water network, also destabilizes m⁶A binding. Lastly, we formulate and test an easy-to-implement approach for increasing the agreement between simulations and NMR experiments by using the HBfix potential function for stabilization of key NOE distances. We call the new approach NOEfix.

Conclusions The structured water molecules surrounding the bound RNA and the methylated substrate’s ability to exclude bulk water molecules are important elements of the YTH domain’s preference for m⁶A. Network of water molecules also fine tunes the specificity towards 5′-Gm⁶A-3′ motifs.

General Significance Our interdisciplinary study of YTHDC1 protein/RNA complex reveals an unusual mechanism by which solvent dynamics can contribute towards recognition of methylation by proteins.