Background: The STE20/SPS1-related proline/alanine-rich kinase (SPAK) is a component of WNK-SPAK/OSR1 signaling pathway that plays an essential role in blood pressure regulation. The function of SPAK is mediated by its highly conserved C-terminal domain (CTD) that interacts with RFXV/I motifs of upstream activators, WNK kinases, and downstream substrate, cation- chloride cotransporters.

Objective: To determine and validate the three-dimensional structure of the CTD of SPAK and to study and analyze its interaction with the RFXV/I motifs.

Methods: A homology model of SPAK CTD was generated and validated through multiple approaches. The model was based on utilizing the OSR1 protein kinase as a template. This model was subjected to a 100 ns molecular dynamic (MD) simulation to evaluate its dynamic stability. The final equilibrated model was used to dock the RFQV-peptide derived from WNK4 into the primary pocket that was determined based on the homology sequence between human SPAK and OSR1 CTDs. The mechanism of interaction, conformational rearrangement and dynamic stability of the binding of RFQV-peptide to SPAK CTD were characterized by molecular docking and molecular dynamic simulation.

Results: The MD simulation suggested that the binding of RFQV induces a large conformational change due to the distribution of salt bridge within the loop regions. These results may help in understanding the relationship between the structure and function of SPAK CTD and to support the drug design of potential SPAK kinase inhibitors as antihypertensive agents.

Conclusion: This study provides deep insight into the SPAK CTD structure and function relationship.

背景：STE20/SPS1 相关的富含脯氨酸/丙氨酸激酶 (SPAK) 是 WNK-SPAK/OSR1 信号通路的一个组成部分，在血压调节中起重要作用。SPAK 的功能由其高度保守的 C 末端结构域 (CTD) 介导，该结构域与上游激活剂 WNK 激酶和下游底物阳离子氯化物协同转运蛋白的 RFXV/I 基序相互作用。

目的：确定和验证SPAK CTD的三维结构，并研究和分析其与RFXV/I基序的相互作用。

方法：通过多种方法生成并验证SPAK CTD的同源模型。该模型基于利用 OSR1 蛋白激酶作为模板。该模型经过 100 ns 分子动力学 (MD) 模拟以评估其动态稳定性。最终的平衡模型用于将源自 WNK4 的 RFQV 肽对接到基于人 SPAK 和 OSR1 CTD 之间的同源序列确定的主要口袋中。通过分子对接和分子动力学模拟表征RFQV-肽与SPAK CTD结合的相互作用机制、构象重排和动态稳定性。

结果：MD 模拟表明，由于环区域内盐桥的分布，RFQV 的结合引起了较大的构象变化。这些结果可能有助于理解 SPAK CTD 的结构和功能之间的关系，并支持潜在的 SPAK 激酶抑制剂作为抗高血压药物的药物设计。

结论：这项研究提供了对 SPAK CTD 结构和功能关系的深入了解。