Dogan Jakob, Jonasson Josefin, Andersson Eva, Jemth Per
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
Biochemistry. 2015 Aug 4;54(30):4741-50. doi: 10.1021/acs.biochem.5b00520. Epub 2015 Jul 20.
Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.
内在无序蛋白（IDP）在蛋白质组中大量存在，并参与关键的细胞功能。然而，关于IDP结合动力学随不同环境条件变化的实验数据却很稀少。我们对CREB结合蛋白的熔球态核共激活因子结合结构域（NCBD）与五种不同蛋白质配体之间相互作用的离子强度依赖性进行了广泛表征，这五种配体包括p160转录共激活因子（SRC1、TIF2、ACTR）的内在无序激活结构域、p53反式激活结构域以及转录因子ETS-2的折叠尖结构域（PNT）。在相同条件下对结合速率常数的直接比较表明，NCBD与无序蛋白结构域之间相互作用的缔合速率常数kon在低盐浓度下较高（4℃时为90 - 350×10⁶ M⁻¹ s⁻¹），但随着离子强度增加而显著降低（降低10 - 30倍），并在生理离子强度附近达到平稳状态。相比之下，NCBD与折叠的PNT结构域之间相互作用的kon在4℃和低盐条件下仅为7×10⁶ M⁻¹ s⁻¹，且离子强度依赖性较弱，这可能反映了一种明显不同的缔合方式，其对静电相互作用的依赖较少。此外，NCBD相互作用的基础速率常数（在不存在静电相互作用时）较高，超过了折叠蛋白通常观察到的数值。一种可能的解释是，无序蛋白有大量可能的碰撞，从而导致形成有成效的沿途径相遇复合物，而折叠蛋白在取向方面受到更多限制。我们的结果突出了静电相互作用在涉及IDP的结合中的重要性，并强调了在比较IDP与有序蛋白结合特性的研究中纳入离子强度作为一个因素的重要性。
