Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, 94025, USA.
Sci Rep. 2018 Nov 12;8(1):16665. doi: 10.1038/s41598-018-34339-1.
Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.
具有多功能调节结构域的蛋白质通常表现出结构的可塑性或蛋白质的无序性，从而允许结合多个调节因子和翻译后修饰。虽然蛋白质无序性的重要性是显而易见的，但它也给体外特性分析带来了挑战。在这里，我们报告了植物分子系统中蛋白质内无序性的情况，尽管这种情况很普遍，但研究却较少。我们对芸薹属植物二酰基甘油酰基转移酶 1（DGAT1）的整个细胞质 N 端结构域进行了详细的生物物理特性分析，其中包括一个抑制模块和别构结合位点。我们的结果表明，单体 N 端结构域可以稳定进行生物物理特性分析，并且在溶液中主要呈无序状态。该结构域在微摩尔浓度下与 DGAT1 的别构调节剂 CoA 和油酰 CoA 相互作用。虽然溶液散射研究表明 DGAT1 的 N 端结构域存在构象异质性，但配体结合会诱导产生少量的二级结构。
