Université de Toulouse, Toulouse, France.
Biophys J. 2010 Oct 6;99(7):2225-34. doi: 10.1016/j.bpj.2010.07.040.
We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.
我们报告了来自解脂耶氏酵母的 Lip2 脂肪酶在其封闭构象下的 1.7 Å 分辨率晶体结构。Lip2 结构与已知的真菌脂肪酶家族（Thermomyces lanuginosa、Rhizopus niveus 和 Rhizomucor miehei 脂肪酶）的结构高度同源。然而，它也呈现出一些独特的特征，这些特征在这里进行了详细的描述和讨论。结构差异，特别是所谓的盖子亚结构域所采用的构象，表明 Lip2 的开启机制可能与其他真菌脂肪酶不同。由于脂肪酶的催化活性强烈依赖于这个可移动亚结构域的结构重排，我们专注于阐明盖子运动的分子机制。使用 Lip2 的 X 射线结构，我们在明确的溶剂环境（水和水/辛烷界面）中进行了广泛的分子动力学模拟，以表征在溶剂或底物结合的影响下盖子经历的主要结构重排。总体而言，我们的结果表明存在一个两步开启机制，首先在蛋白质吸附在水/有机溶剂界面时产生半开构象，然后在底物结合时进一步打开盖子。
