Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds

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是一种二聚体丝氨酸/苏氨酸蛋白激酶，属于NIMA相关激酶（NIMA-related kinases, Neks）家族。其N端催化结构域与C端调节区域由亮氨酸拉链（leucine zipper）相连，该结构域对Nek2催化活性的激活具有关键作用。此前，利用X射线晶体学与核磁共振（Nuclear Magnetic Resonance, NMR）技术解析Nek2亮氨酸拉链结构的所有尝试，均因该区域存在异常的中等/慢速时间尺度构象动态变化而未能成功。研究人员采用二硫键工程（disulfide engineering）策略，即在卷曲螺旋（coiled coil）侧翼的柔性区域引入非天然二硫键，以调控这一重要二聚化结构域的构象交换动态。由此带来的交换速率降低，使核磁共振光谱中的关键特征得到显著改善，包括谱线宽度、相干转移泄漏与弛豫现象。研究团队针对野生型蛋白、两种携带单二硫键的突变体以及一种携带双二硫键的突变体，全面分析了上述效应。此外，研究人员在宽温度范围内测定了交换动力学参数，得以详细解析活化能（ΔG‡）与最大速率常数（k’ex）。针对一种在C端携带二硫键的突变体，研究人员成功为两种构象异构体完成了完整的主链核磁共振归属，证实了二硫键工程的应用优势。本研究首次成功将"动力学型"二硫键应用于调控蛋白质动态变化带来的不利影响。其一，该工作为未来全面解析Nek2亮氨酸拉链的结构与功能提供了极具潜力的可靠研究平台；其二，该研究拓展了二硫键蛋白工程的工具箱——除已广泛应用的热力学用途外，新增了动力学调控这一全新途径。