Laboratory of Chemistry and Computational Chemistry, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne EPFL, 1015 Lausanne, Switzerland.
Chembiochem. 2013 Apr 15;14(6):703-10. doi: 10.1002/cbic.201200566. Epub 2013 Mar 26.
O(6) -alkylguanine-DNA alkyltransferase (AGT) adopts a non-enzymatic suicide mechanism for the repair of methylated guanine bases by transferring the methyl adduct to itself, thereby initiating unfolding and fast degradation. Classical molecular dynamics simulations provide quantitative evidence that two conserved glycine residues at the centre of an α-helix make the structure susceptible to structural perturbations. The stability of this helix, designated the "recognition helix", is an important factor during the early onset of unfolding of human AGT (hAGT). By combining theory and experiment, we found that helical stability is controlled by key factors in the surrounding protein structure. By using a "double-clip" mechanism, nearby residues hydrogen bond to both the base and centre of the helix. This double clip stabilises this site in the protein in the absence of substrate, but the helix is destabilised upon alkylation. The present investigation aimed to establish why alkylation of hAGT leads to conformational changes and how the protein environment functions as a switch, thus turning the stability of the protein "on" or "off" to tune degradability.
O(6)-烷基鸟嘌呤-DNA 烷基转移酶（AGT）通过将甲基加合物转移到自身，采用非酶自杀机制来修复甲基化鸟嘌呤碱基，从而引发解折叠和快速降解。经典分子动力学模拟为两个保守的甘氨酸残基在α-螺旋中心使结构易受结构干扰提供了定量证据。该螺旋的稳定性，称为“识别螺旋”，是人类 AGT（hAGT）早期解折叠的重要因素。通过结合理论和实验，我们发现螺旋稳定性受周围蛋白质结构中关键因素的控制。通过使用“双夹”机制，附近的残基与螺旋的碱基和中心形成氢键。在没有底物的情况下，这种双夹稳定了该蛋白质中的该位点，但烷基化会使螺旋不稳定。本研究旨在确定为什么 hAGT 的烷基化会导致构象变化，以及蛋白质环境如何作为开关起作用，从而使蛋白质的稳定性“开启”或“关闭”，以调节降解性。
