Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy.
CEINGE Advanced Biotechnologies, Via G. Salvatore 486, 80145 Naples, Italy.
Int J Mol Sci. 2024 Aug 15;25(16):8891. doi: 10.3390/ijms25168891.
Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction.
周期素依赖性激酶样 5（CDKL5）是一种丝氨酸/苏氨酸蛋白激酶，参与人类大脑的发育和功能。CDKL5 中的突变，特别是其催化结构域的突变，会导致一种严重的发育障碍，称为 CDKL5 缺乏症。然而，对于这些突变所导致的结构后果的分子研究仍然缺乏。CDKL5 的催化结构域具有不同的翻译后修饰位点，如磷酸化，但它们在催化活性、蛋白质折叠和稳定性中的作用尚未完全研究。在这项工作中，我们描述了 CDKL5 催化结构域在 中的表达模式，表明它主要聚集。然而，使用可溶性标签、降低表达温度、手动密码子优化以克服内部翻译起始以及将蛋白质与 K 和 MgATP 孵育，可以收集到可溶性的具有催化活性的激酶。有趣的是，与真核对应物相比，所得蛋白质表现出低磷酸化，证明细菌是获得几乎未修饰的 CDKL5 的有用工具。这项研究对 CDKL5 的自动激活机制和稳定性决定因素提出了质疑，为细菌和真核表达蛋白之间的比较生物物理研究提供了有价值的平台，有助于我们理解与 CDKL5 功能障碍相关的神经发育障碍。
