MD trajectories for "Kinetic barrier to enzyme inhibition is manipulated by dynamical local interactions in E. coli DHFR"

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model<br> protein for understanding enzyme dynamics. DHFR’s crucial role in folate synthesis renders it<br> an ideal candidate to understand protein function and protein evolution mechanisms. In this<br> study, to understand how a newly proposed DHFR inhibitor, 4’-deoxy methyl trimethoprim<br> (4’-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior<br> performance over trimethoprim (TMP). To elucidate the inhibition mechanism of 4’-DTMP,<br> we first confirmed, both computationally and experimentally, that the relative binding free<br> energy cost for the mutation of TMP and 4’-DTMP are the same, pointing to the origin of the<br> characteristic differences to be kinetic rather than thermodynamic. We then employed an<br> interaction-based analysis by focusing first on the active site, then on the whole enzyme. We<br> confirmed that the polar modification in 4’-DTMP induces additional local interactions with<br> the enzyme, particularly the M20 loop. These changes are propagated to the whole enzyme as<br> shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support<br> our analysis with network-based community analysis and show that segmentation of the loop<br> domain of the inhibitor-bound DHFR must be avoided by a successful inhibitor.二氢叶酸还原酶（Dihydrofolate reductase, DHFR）是一类重要的药物靶点，同时也是用于解析酶动力学特性的经典研究模型蛋白。DHFR在叶酸合成通路中的关键作用，使其成为阐释蛋白质功能与蛋白质进化机制的理想研究对象。 本研究旨在探究新型DHFR抑制剂4’-脱氧甲基甲氧苄啶（4’-deoxy methyl trimethoprim, 4’-DTMP）如何改变进化轨迹，为此对其相较于甲氧苄啶（trimethoprim, TMP）更优异的性能背后的相互作用机制展开了研究。 为阐明4’-DTMP的抑制机制，本研究首先通过计算与实验相结合的手段，证实TMP与4’-DTMP的突变相对结合自由能成本一致，这表明二者的特性差异源于动力学层面而非热力学层面。 随后我们采用基于相互作用的分析策略，先聚焦于酶的活性位点，再扩展至整个酶分子。 研究证实，4’-DTMP上的极性修饰可诱导其与酶，尤其是M20环，产生额外的局部相互作用。这类变化会通过氢键网络的改变传递至整个酶体。 为解析变构相互作用机制，本研究通过基于网络的群落分析对上述分析进行佐证，并证实：一款成功的抑制剂必须避免造成抑制剂结合态DHFR的环结构域发生片段化。