Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK

Abstract

The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was determined in the unphosphorylated inactive form and, like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK and OSR1, we have determined the crystal structures of SPAK 63-403 at 3.1 Å and SPAK 63-390 T243D at 2.5 Å resolution. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without and the shorter with an activating T243D point mutation. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1 but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1 but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of partially active SPAK T243D is consistent with a multistage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.

Figure 1. Domain organization and structure of SPAK WT and T243D

(A) Domain organization of mouse SPAK. The kinase domain (residue 75–350) is shown in blue, the PF1 domain (residues 350–403) in green, and the PF2 domain (residue 462–556) in darker green. The SPAK T243D fragment crystallized is boxed. (B) Activation loop domain-swapped dimers of SPAK T243D and SPAK WT. The two SPAK T243D dimer subunits are colored orange and blue, and the two SPAK WT subunits are colored purple and yellow. (C) Ribbon diagram of a SPAK T243D subunit (or monomer) as observed in the domain-swapped dimer. α-helices-cyan; β-strands-magenta; glycine-rich loop- brown; catalytic loop- red; activation loop- blue; PF1 domain- yellow. AMP-PNP is shown in a stick representation.

Figure 2. Conformational changes occurring in SPAK T243D compared to SPAK WT and OSR1 WT

(A) Cartoon representation (α-helices represented as cylinders for clarity) of C-terminal domain alignments of SPAK WT (chain A; purple), SPAK T243D (chain A; orange), and OSR1 WT (chain A; green; PDB: 3DAK). Structural elements of particular interest are labeled. (B) Dimer interface near the domain-swapped activation loop and SPAK T243/OSR1 T185 WNK phosphorylation site. Labels marked with a prime (‘) correspond to the second monomer in the dimer. Spheres represent the first residue observed in the activation loop electron density and the SPAK T247'/OSR1 T189' catalytic residue. (C) The D-K-T catalytic triad interaction, formed by catalytic loop residues D204 and K206 and domain-swapped activation loop residue T247', is only present is SPAK T243D. (D) The essential K-E ion pair, between β3 K104 and αC E121, which is a hallmark of active kinases, is not present in SPAK WT or SPAK T243D. An interaction between catalytic loop residue R203 and E121 is present in SPAK WT but lost in SPAK T243D.

Figure 3. Identification and mutation of the domain-swap hinge

(A) Multiple sequence alignment of representative sequences of domain swapped kinases SPAK, DAPK3, CHK2, SLK, LOK, compared to the non-swapped kinases PAK6 and TAO2. The position corresponding to G261 in SPAK is boxed in green. (B) Superposition of SPAK T243D and the non-swapped kinase PAK6 (PDB 2C30) reveals the difference in the αEF/αF loop. SPAK is colored in orange and PAK6 is in gray. The switch point R260/G261 in SPAK and S576/L577 in PAK6 are represented in sticks and colored in red and purple, respectively. (C) View near αEF and αEF’ helices using chain A structural alignments of SPAK WT, SPAK T243D, and OSR1 WT dimers. Positional differences begin to occur near G261 and G261' (G203 in OSR1). (D) Gel filtration profile of SPAK T243D and SPAK (T243D/R260S/G261L).

Figure 4. Effect of the R260S/G261L mutation on SPAK activity

(A) Coupled protein kinase assay. SPAK 63–403 WT and R260S/G261L mutant were preincubated with ATP-MgCl₂ for 30 minutes with or without WNK1, after which [γ-³²P]-ATP was added for radiolabeling, and GST-NKCC2 1–175 was added as substrate. (B) Kinase assays comparing activity of SPAK 63–403 with and without the R260S/G261L mutation. T243E is an activating phosphomimetic mutation in the SPAK activation loop. All results in (A) and (B) are reported as relative activities, and as the mean and standard deviation of triplicate measurements quantified by phosphoimaging.