Randall Centre and School of Cardiovascular Medicine and Sciences, King's College London, London, United Kingodm.
The Henry Wellcome Building for Biomolecular NMR Spectroscopy, Institute of Cancer & Genomic Sciences, University of Birmingham, Birmingham, United Kingodm.
PLoS One. 2019 Feb 1;14(2):e0210352. doi: 10.1371/journal.pone.0210352. eCollection 2019.
Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2's catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG‡) and maximal rate constant (k'ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of 'kinetic' disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.
Nek2 是一种二聚丝氨酸/苏氨酸蛋白激酶，属于 NIMA 相关激酶（Neks）家族。其 N 端催化结构域和 C 端调节区由亮氨酸拉链桥接，该拉链在 Nek2 催化活性的激活中起着重要作用。异常的中间/慢时变构动态迄今为止已挫败了通过 X 射线晶体学和核磁共振（NMR）来确定 Nek2 亮氨酸拉链结构的所有尝试。二硫键工程是将非天然二硫键战略性地置于卷曲螺旋侧翼的柔性区域中，用于调节该重要二聚化结构域的构象交换动力学。交换速率的降低导致 NMR 光谱中重要特征的实质性改进，例如线宽、相干转移泄漏和弛豫。对于野生型蛋白、两个单二硫键突变体和另一个双二硫键携带突变体，全面分析了这些效应。此外，在很宽的温度范围内测量了交换动力学，允许对活化能（ΔG‡）和最大速率常数（k'ex）进行详细分析。对于在 C 端带有二硫键的一个突变体，可以获得两种构象的完整骨架 NMR 分配，证明了二硫键工程的好处。我们的研究首次成功应用“动力学”二硫键来控制蛋白质动力学的不利影响。首先，这为 Nek2 亮氨酸拉链的未来全面结构和功能研究提供了一个有前途的、稳健的平台。其次，通过除了已建立的二硫键热力学用途之外增加动力学选项，这项工作通过二硫键扩展了蛋白质工程的工具包。
