The crystal structure of pseudokinase PEAK1 (Sugen kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization

Abstract

The pseudokinase group encompasses some 10% of protein kinases, but pseudokinases diverge from canonical kinases in key motifs. The two members of the small new kinase family 3 (NKF3) group are considered pseudokinases. These proteins, pseudopodium-enriched atypical kinase 1 (PEAK1, Sugen kinase 269, or SgK269) and pragmin (Sugen kinase 223 or SgK223), act as scaffolds in growth factor signaling pathways, and both contain a kinase fold with degraded kinase motifs at their C termini. These kinases may harbor regions that mediate oligomerization or control other aspects of signal transduction, but a lack of structural information has precluded detailed investigations into their functional roles. In this study, we determined the X-ray crystal structure of the PEAK1 pseudokinase domain to 2.3 Å resolution. The structure revealed that the PEAK1 kinase-like domain contains a closed nucleotide-binding cleft that in this conformation may deleteriously affect nucleotide binding. Moreover, we found that N- and C-terminal extensions create a highly unusual all α-helical split-dimerization region, termed here the split helical dimerization (SHED) region. Sequence conservation analysis suggested that this region facilitates a dimerization mode that is conserved between PEAK1 and pragmin. Finally, we observed structural similarities between the PEAK1 SHED region and the C-terminal extension of the Parkinson's disease-associated kinase PINK1. In summary, PEAK1's kinase cleft is occluded, and its newly identified SHED region may promote an unexpected dimerization mode. Similarities of PEAK1 with the active kinase PINK1 may reclassify the latter as a member of the new kinase family 3 group.

Keywords: PTEN-induced putative kinase (PINK1); crystal structure; dimerization; protein kinase; signal transduction.

Figure 1.

PEAK1 domains and kinase motifs. A, schematic diagram of PEAK1. Regions and domains are shown. The PEAK1 pseudokinase domain and SHED region are indicated. Some of the PEAK1 phosphorylation sites are noted, with prominent phosphotyrosine-binding proteins indicated, e.g. the Src SH2 binding site at Tyr⁶⁶⁵ (pYEEI). B, PEAK1 kinase motifs. Consensus shows consensus sequences of kinase fold motifs. δ indicates hydrophilic residue, and ϕ indicates hydrophobic residue. hPEAK1 indicates sequence of human PEAK1 kinase fold motifs. Residues that strictly conform to the consensus are colored red. 111 sequences indicates consensus of PEAK1 motifs across 111 sequences; *, completely conserved; :, strongly similar properties; ·, weakly similar properties by Clustal Omega (40).

Figure 2.

Structure of PEAK1 pseudokinase. A, structure of PEAK1 pseudokinase domain. The cartoon format is shown with the α-helices and β-strands indicated. Kinase N-lobe is colored light gray, and the C-lobe is in dark gray. The β4–β5 insert is shown in red. SHED domain colored green. Unmodeled loops are indicated by dashed lines. B and C, example electron density maps for two regions of the structure, the glycine-rich loop (B), and helices αS and αK (C). The representative residue numbers are indicated. Water molecules shown as red spheres. 2F_obs − F_calc map: blue shows 1σ, and light blue shows 2σ. In the F_obs − F_calc map, green shows 3σ, and red shows −3σ. D, DSSP-defined (41) topology map of PEAK1 pseudokinase domain. Secondary structure elements are indicated with α-helices as cylinders, β-strands as arrows, and 3₁₀ helices as squiggles. The SHED domain is colored green. β4–β5 insert shown in red. Unbuilt loops are indicated in gray/pale red. E, ATP cleft of PEAK1. Residues of the glycine-rich loop are shown in blue, the activation segment is in pink (including the DFG/NFS motif), the HRDXKXXN (HCDLRLEN) motif is in yellow, and the β4–β5 insert in red. Hydrogen-bonding interactions that seem to stabilize the glycine-rich loop are shown as green dashed lines. F, hydrophobic spines of PEAK1 pseudokinase domain. The regulatory spine is shaded in green, and the catalytic spine is in yellow. Tyr¹³⁴³ completes the catalytic spine.

Figure 3.

PEAK1 SHED region. A, PEAK1 homodimer. Symmetry mates shown for the PEAK1 homodimer, colored and labeled as per Fig. 2A. The secondary structure of the SHED region is indicated. Surface of one copy is shown. B, map of dimerization interactions. Electrostatic interactions are shown as red dashed lines, van der Waals interactions are shown as straight lines. The helices are indicated. Residue Ala¹⁷⁰⁷ is indicated in red. C, close-up view of the dimerization interaction. Residues that interact are indicated. Residue Ala¹⁷⁰⁷ is indicated in red. D, electrostatic potential of the interaction interface. Electropositive potential is shown in blue, and electronegative potential is in red. Helices αS and αK are shown in green. E, conservation of the interaction interface over 111 sequences of PEAK1. Complete conservation is shown in blue. Low conservation is in white. The results were calculated by Consurf server (42). Helices αS and αK are shown in green. F, size exclusion chromatography for wildtype and A1707D mutant PEAK1. Incorporation of the single point mutation significantly lengthens the retention time on a Superdex 200 Increase 10/300 GL (GE) column, suggesting that the A1707D mutant is a monomer.

Figure 4.

Comparison of PEAK1 and pragmin. A, alignment of PEAK1 and pragmin. DSSP-defined (41) secondary structure elements for PEAK1 are shown, with the α-helices as cylinders, β-strands as arrows, and 3₁₀ helices as squiggles. Helices S′, 4′, and E′ are 3₁₀. Build residues of PEAK1 are in bold type. Conserved kinase motifs are indicated in the Motifs rows. Residues of PEAK1 that interact in the SHED region homodimerization interaction are indicated in the Interact rows. Conservation was calculated by Clustal Omega (40) and indicated as completely conserved (*), strongly similar properties (:), or weakly similar properties (·). B, conservation of the interaction interface between PEAK1 and pragmin. Complete conservation is shown in blue. Low conservation is in white.

Figure 5.

Comparison of the structures PEAK1 and PINK1. A, structure of PINK1 (PDB code 5OAT) (29) showing crystallographic CTE–mediated dimerization via a symmetry mate. Kinase domains are in yellow, and CTEs are in blue. B, superposition of PINK1 CTE and one-half of the PEAK1 SHED region illustrating the similarity of the regions. The helices of PEAK1 are indicated and shown in green. C, top view of superposed PINK1 CTE and one-half of the PEAK1 SHED region.

Figure 6.

Occlusion of ATP-binding clefts in pseudokinases. A, example of an active LIMK1 bound to ATP analogue AMP-PNP (PDB code 5HVK; Ref. 43). B, PEAK1 showing Tyr¹³⁴³ in the ATP-binding cleft. C, MviN pseudokinase showing residues Phe⁷²⁴, Trp⁷⁸⁹, and Tyr⁸⁴⁹ occluding the ATP binding cleft (PDB code 3OTV; Ref. 32). D, Ror2 showing Tyr⁵⁵⁵ occluding the ATP-binding cleft (PDB code 4GT4; Ref. .33). Structures are superposed on the C-lobes to show the same orientation.