Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-1068, USA.
Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Mol Biol. 2021 Sep 3;433(18):167122. doi: 10.1016/j.jmb.2021.167122. Epub 2021 Jun 25.
Highly negatively charged segments containing only aspartate or glutamate residues ("D/E repeats") are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40-900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.
富含天冬氨酸或谷氨酸残基（"D/E 重复"）的高度带负电荷片段存在于许多真核生物蛋白中。例如，HMGB1 蛋白的 C 端 30 个残基完全是 D/E 重复。我们使用核磁共振（NMR）、荧光和计算方法研究了 D/E 重复如何导致 HMGB1 自身抑制其与顺铂修饰的 DNA 的特异性结合。通过在很宽的离子强度范围内（40-900 mM）改变离子强度，我们能够将自身抑制和非抑制状态之间的构象平衡向任一侧完全移动。这使我们能够在各种离子强度下确定 HMGB1 自身抑制的宏观和微观平衡常数。在宏观水平上，涉及自身抑制和非抑制状态的模型可以解释盐浓度依赖性结合亲和力数据。我们在微观水平上的数据表明，D/E 重复和 HMGB1 的其他部分经历静电模糊相互作用，每个相互作用都比宏观自身抑制效应所预期的要弱。这种差异表明，模糊相互作用的多价性质使得在宏观水平上能够进行强烈的自身抑制，尽管每个位点的分子内相互作用相对较弱。实验和计算数据均表明，D/E 重复优先与 HMGB1 的其他固有无序区域（IDR）相互作用。我们还发现，模拟与 HMGB1 核输出相关的翻译后修饰的突变可以适度调节 DNA 结合亲和力，可能通过影响自身抑制来实现。这项研究阐明了 D/E 重复的模糊相互作用的功能作用。
