Department of Chemistry, UNC-Chapel Hill, Chapel Hill, North Carolina, USA; Molecular and Cellular Biophysics Program, UNC-Chapel Hill, Chapel Hill, North Carolina, USA.
School of Pharmacy, University College London, London, United Kingdom.
J Biol Chem. 2023 Mar;299(3):102984. doi: 10.1016/j.jbc.2023.102984. Epub 2023 Feb 3.
Biophysical characterization of protein-protein interactions involving disordered proteins is challenging. A common simplification is to measure the thermodynamics and kinetics of disordered site binding using peptides containing only the minimum residues necessary. We should not assume, however, that these few residues tell the whole story. Son of sevenless, a multidomain signaling protein from Drosophila melanogaster, is critical to the mitogen-activated protein kinase pathway, passing an external signal to Ras, which leads to cellular responses. The disordered 55 kDa C-terminal domain of Son of sevenless is an autoinhibitor that blocks guanidine exchange factor activity. Activation requires another protein, Downstream of receptor kinase (Drk), which contains two Src homology 3 domains. Here, we utilized NMR spectroscopy and isothermal titration calorimetry to quantify the thermodynamics and kinetics of the N-terminal Src homology 3 domain binding to the strongest sites incorporated into the flanking disordered sequences. Comparing these results to those for isolated peptides provides information about how the larger domain affects binding. The affinities of sites on the disordered domain are like those of the peptides at low temperatures but less sensitive to temperature. Our results, combined with observations showing that intrinsically disordered proteins become more compact with increasing temperature, suggest a mechanism for this effect.
生物物理特性的蛋白质 - 蛋白质相互作用涉及无规卷曲蛋白质是具有挑战性的。一个常见的简化是使用只包含最小必需残基的肽来测量无规位点结合的热力学和动力学。然而，我们不应假设这些少数残基就能说明全部情况。来自黑腹果蝇的多结构域信号蛋白 Son of sevenless 对于丝裂原活化蛋白激酶途径至关重要，它将外部信号传递给 Ras，从而导致细胞反应。无规卷曲的 55 kDa C 末端结构域是一种自抑制因子，可阻止鸟嘌呤核苷酸交换因子的活性。激活需要另一种蛋白质，受体激酶下游蛋白（Drk），它包含两个Src 同源 3 结构域。在这里，我们利用 NMR 光谱和等温热滴定法来量化 N 端 Src 同源 3 结构域与融合到无规序列侧翼的最强结合位点的热力学和动力学。将这些结果与孤立肽的结果进行比较，可以提供有关较大结构域如何影响结合的信息。无序域上的结合位点的亲和力与肽在低温下的亲和力相似，但对温度的敏感性较低。我们的结果结合表明，随着温度的升高，固有无序蛋白变得更加紧凑，这表明了这种效应的机制。
