Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
J Chem Theory Comput. 2021 Feb 9;17(2):1240-1249. doi: 10.1021/acs.jctc.0c01136. Epub 2021 Jan 20.
N6-Methyladenosine (mA) 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 mA recognition by the reader proteins is still elusive. Here, we investigate this recognition process by combining atomistic simulations, site-directed mutagenesis, and biophysical experiments using YTHDC1 as a model. We find that the N6 methyl group of mA 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 mA-containing RNA in solution. The mA binding site dynamically equilibrates between multiple metastable conformations with four residues being involved in the regulation of mA 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 mA 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 mA 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, mA-editing tools, and drug candidates against reader proteins.
N6-甲基腺苷（mA）是真核信使 RNA（mRNA）中最常见的修饰，其细胞加工和功能受读者蛋白 YTHDC 和 YTHDF 的调节。然而，读者蛋白识别 mA 的机制仍不清楚。在这里，我们以 YTHDC1 为模型，通过结合原子模拟、定点突变和生物物理实验来研究这个识别过程。我们发现，mA 的 N6 甲基通过其与芳香族笼（由 Trp377 和 Trp428 形成）的特异性相互作用以及有利于 mA 含有 RNA 在溶液中易于缔合的构象来促进结合。mA 结合位点在多个亚稳定构象之间动态平衡，其中四个残基参与 mA 结合的调节（Trp428、Met438、Ser378 和 Thr379）。Trp428 在构建和拆除芳香族笼之间切换两种构象状态。有趣的是，将 Met438 和 Ser378 突变为丙氨酸不会改变 mA 与蛋白质的结合，但会显著重新分配结合焓和熵项，即焓熵补偿。这种补偿是由于野生型和突变蛋白的构象变化以及水分子的重新分布所伴随的不同熵-焓传递所致。相比之下，点突变 Thr379Val 会显著改变 YTHDC1 与天然配体的热稳定性和结合能力。此外，热力学分析和自由能计算揭示了一个结构水分子的作用，该水分子与 mA 协同结合 YTHDC1，并作为氢键网络的中心。综上所述，实验数据和模拟结果可能会加速化学探针、mA 编辑工具和针对读者蛋白的药物候选物的发现。
