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
protein for understanding enzyme dynamics. DHFR’s crucial role in folate synthesis renders it
an ideal candidate to understand protein function and protein evolution mechanisms. In this
study, to understand how a newly proposed DHFR inhibitor, 4’-deoxy methyl trimethoprim
(4’-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior
performance over trimethoprim (TMP). To elucidate the inhibition mechanism of 4’-DTMP,
we first confirmed, both computationally and experimentally, that the relative binding free
energy cost for the mutation of TMP and 4’-DTMP are the same, pointing to the origin of the
characteristic differences to be kinetic rather than thermodynamic. We then employed an
interaction-based analysis by focusing first on the active site, then on the whole enzyme. We
confirmed that the polar modification in 4’-DTMP induces additional local interactions with
the enzyme, particularly the M20 loop. These changes are propagated to the whole enzyme as
shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support
our analysis with network-based community analysis and show that segmentation of the loop
domain of the inhibitor-bound DHFR must be avoided by a successful inhibitor.

二氢叶酸还原酶（DHFR）是一类重要的药物靶点，同时也是用于解析酶动力学机制的经典研究模型蛋白。DHFR在叶酸合成通路中发挥关键作用，使其成为探究蛋白质功能与演化机制的理想研究对象。本研究为探究新型DHFR抑制剂4’-脱氧甲基甲氧苄啶（4’-DTMP）如何改变蛋白质演化轨迹，针对其相较于甲氧苄啶（TMP）更优异的作用机制展开了相互作用层面的研究。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方式证实：TMP与4’-DTMP发生突变时的相对结合自由能成本一致，这表明二者功能特性的差异源于动力学层面，而非热力学层面。随后，本研究采用基于相互作用的分析策略，先聚焦酶的活性位点，再拓展至整个酶分子。研究证实，4’-DTMP分子上的极性修饰基团可诱导其与酶产生额外的局部相互作用，尤其是与M20环。这些变化会通过氢键网络的改变传递至整个酶分子。为进一步揭示别构相互作用机制，本研究借助基于网络的群落分析验证了分析结果，并证实：一款有效的抑制剂必须避免引发与抑制剂结合的DHFR的环结构域发生结构割裂。