Background: The STE20/SPS1-related proline/alanine-rich kinase (SPAK) is a component of WNKSPAK/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 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 relation between the
structure and function of SPAK CTD and to support drug design of potential SPAK kinase inhibitors as antihypertensive
Conclusion: This study provides deep insight into SPAK CTD structure and function relationship.