Laboratoire des Biomolécules, Département de chimie, École normale supérieure, UPMC Université Paris 06, CNRS, PSL Research University , 24 rue Lhomond, Paris 75005, France.
Sorbonne Universités, UPMC Université Paris 06, École normale supérieure, CNRS, Laboratoire des Biomolécules (LBM) , Paris 75005, France.
J Am Chem Soc. 2017 Sep 6;139(35):12219-12227. doi: 10.1021/jacs.7b05823. Epub 2017 Aug 28.
Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.
许多无规则卷曲蛋白质（IDP）和蛋白质区域（IDR）与各种蛋白质伴侣发生短暂但特异性的相互作用。通常（如果不是总是如此），与蛋白质伴侣的相互作用会导致 IDR 的部分折叠。描述此类复合物的构象空间具有挑战性：在溶液 NMR 中，相互作用区域中 IDR 的信号变得广泛、微弱，并且常常不可见，而 X 射线晶体学仅提供完全有序区域的信息。因此，需要一种简单的方法来描述 IDP 结合状态下完全和部分有序区域。在这里，我们介绍了一种基于 NMR 监测化学交换的方法，通过观察蛋白质的自由状态来研究结合后折叠的 IDR 状态。结合状态的结构约束来自化学位移，并且通过弛豫率来表征结合状态的位点特异性动力学。确定 IDR 的相互作用部分的构象，随后将其对接至折叠伴侣的结构上。我们将该方法应用于研究 Artemis 无规则卷曲 C 端区域与 Ligase IV DNA 结合域之间的相互作用。我们表明，我们可以准确地重现 X 射线晶体学确定的复合物核心结构，并确定更广泛的界面。该方法广泛适用于无序蛋白质和折叠蛋白质复合物的生物物理研究。
