Structural Analysis of STE20-Related Proline-Alanine-Rich Kinase (SPAK)
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Ste20-related proline-alanine-rich kinase (SPAK) is a kinase that regulates ion cotransporters including KCC, NKCC1, NKCC2 and NCC. Recently, SPAK was identified as a target regulated by the WNK (With No lysine (K)) family of protein kinases. Overexpression of WNK1 has been associated to a hereditary form of hypertension. The link between WNK-SPAK-cotransporters provides one of the functional pathways for WNK to regulate intracellular and extracellular salt and water balance, thus contribute to the control of blood pressure. My study focused on determining the crystal structure of SPAK and providing a molecular basis for understanding the catalytic and regulatory mechanisms of this enzyme. I have solved the crystal structure of a partially active form of SPAK 63-390 (T243D). The structure contains the kinase domain and part of the C-terminal PF1 domain with a mutation of the WNK phosphorylation site, Thr243, to aspartate to mimic the phosphorylated state. The structure reveals two dimer interfaces. One is from the unique activation loop-swapped structure. The other is formed between the N-terminal domain of one molecule and the C-terminal domain of the second molecule. Comparison the structure with unphosphorylated inactive OSR1 revealed significant conformational changes in the glycine-rich loop, helix aC, and the activation loop. A more striking feature is the reformation of the P+1 pocket in SPAK. The interaction is formed by residues from both molecules of the domain-swapped dimer supporting the conclusion that the domain-swapped dimer is a functional unit. As remodeling of the P+1 pocket is usually associated with kinase activation, the conformational change found between the crystallized forms of OSR1 and SPAK is thus believed to be a regulated event. The unique activation loop-swapped feature suggests a novel regulatory mechanism that could be used in kinases.