Long-range electrostatic interactions contribute to the target specificity and reactivity of thioredoxin family proteins

蛋白质硫醇的时空还原和氧化是所有生命领域信号转导的重要机制。硫氧还蛋白（Trx）家族蛋白能有效催化硫醇-二硫交换反应，并因其在硫醇开关操作中的重要性而得到广泛认可。Trx家族蛋白具有广泛的同时又非常明显的底物特异性，这是氧化还原开关的先决条件。尽管多方努力，这种特异性的基础仍不清楚。我们之前的工作表明，热力学参数，如氧化还原电位，不能决定氧化还原素的特异性和反应性。相反，催化效率与氧化还原素的特定静电场模式密切相关[化学]。科学学报，2015,(6):7049-7058。有效的反应速率依赖于足够低的活化能垒和高频率的有效碰撞。因此，我们提出了以下模型：(1)远距离识别蛋白质及其适当的（预）取向，由远程静电相互作用主导。(2)蛋白质相互吸引。(3)直接的短程分子相互作用，导致相遇络合物的形成。(4)硫醇-二硫化物交换反应，受到许多热力学限制。(5)配合物的解离。该项目的主要目的是确定特定静电相互作用在反应早期阶段（1-2）的重要性。最重要的是，我们的目的是确定这些力对反应的特异性和总速率常数的贡献。我们研究计划的主要重点是由计算分析和预测指导的蛋白质相互作用的生化和生物物理特征。作为模型，我们建议通过Grxs分析大肠杆菌3'-磷酸腺苷-5'-硫酸磷酸（PAPS）还原酶中的催化二硫化物，人类崩溃反应介质蛋白22 （CRMP2）的氧化还原开关，并与SPP 1710联盟的其他成员合作，分析其他感兴趣的硫醇开关。我们建议用实验确定或模拟的结构来计算蛋白质的静电特性。在此指导下，我们将通过酶动力学、荧光猝灭光谱、表面等离子体共振和原子力光谱分析相互作用。我们将确定关联速率对总速率常数的贡献。此外，我们的目标是设计特定的相互作用，例如，将大肠杆菌Grx3转化为PAPS还原酶的高效还原剂，并优化Grxs与roGFP氧化还原传感器之间的电子传递。毫无疑问，控制Trx家族蛋白底物特异性的机制是理解硫醇转换的关键。

The spatio-temporal reduction and oxidation of protein thiols is an essential mechanism in signal transduction in all kingdoms of life. Thioredoxin (Trx) family proteins efficiently catalyse thiol-disulphide exchange reactions and the proteins are widely recognized for their importance in the operation of thiol switches. Trx family proteins have a broad and at the same time very distinct substrate specificity - a prerequisite for redox switching. Despite of multiple efforts, the basis for this specificity is still unclear. Our previous work suggests that thermodynamic parameters, such as the redox potential, do not determine specificity nor reactivity of the redoxins. Instead, the catalytic efficiency correlated strongly to a specific electrostatic field pattern of the redoxins [Chem. Sci. 6:7049-7058, 2015]. Efficient reaction rates rely on a sufficiently low activation energy barrier and a high frequency of effective collisions. Hence, we propose the following model: (1) The recognition of the proteins from a distance and their proper (pre-)orientation, dominated by long-range electrostatic interactions. (2) Attraction of the proteins towards each other. (3) Direct short-range molecular interactions, leading to the formation of the encounter complex. (4) The thiol-disulphide exchange reaction, subjected to a number of thermodynamic restrictions. (5) The dissociation of the complex. The primary aim of this project is to determine the importance of specific electrostatic interactions in the early phases (1-2) of the reaction. Most of all, we aim to establish the contributions of these forces to the specificity and overall rate constants of the reaction. The major focus of our research programme is the biochemical and biophysical characterisation of protein-protein interactions guided by computational analyses and predictions. As models, we propose to analyse the catalytic disulphide in E coli 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase by Grxs, the redox switch in human collapsin response mediator protein 2 2 (CRMP2) and, in cooperation with other members of the SPP 1710 consortium, other thiol switches of interest. We suggest to compute the electrostatic properties of the proteins using experimentally determined or modelled structures. Guided by these, we will analyse the interactions by enzyme kinetics, fluorescence quenching spectroscopy, surface plasmon resonance, and atomic force spectroscopy. We will determine the contribution of the association rates to the overall rate constants. Moreover, we aim to engineer specific interactions, for instance to turn E. coli Grx3 into an efficient reductant for PAPS reductase and to optimise the electron transfer between Grxs and the roGFP redox sensor. We have no doubt that the mechanisms that control the substrate specificity of Trx family proteins are key to the understanding of thiol switching.