Fig. 3
DRIK1 is a pseudokinase that is unable to bind nucleotides. aZmDRIK1 protein domain structure and multiple amino acid sequence alignment of ZmDRIK1 with a set of active and inactive RLK/Pelle-LRRs. The schematic ZmDRIK1 domain structure shows the position of the low complexity region (green), the transmembrane domain (red) and the kinase domain (blue). Numbers indicate amino acid positions along the protein sequence. The multiple amino acid sequence alignment shows the core of the kinase domains of: DRIK1 from maize, sorghum, rice and Arabidopsis; BIR2, BRI1 and BSK8 and CLV1 from Arabidopsis; IRAK4 from Homo sapiens; PKA from Mus musculus. Conserved residues, essential for kinase activity, are marked in red. Highlighted in yellow are the conserved residues representing the consensus kinase sequence. Secondary structures (β-strands in light blue arrow; α-helix in orange cylinders) are named according to the PKA structure, except αE6, which in PKA is a β-strand 6. b Purification of the ZmDRIK1 kinase domain. ZmDRIK1-KDR187-S514 was cloned into pNIC28a-Bsa4 and expressed in E coli BL21(DE3) pRARE. Lanes are TL, total lysate; S, supernatant; FT, flow through; W, washed out; E, eluted fraction, T+, TEV protease treated. PP, gel filtration purified ZmDRIK1-KDR187-S514. MW, molecular weight of protein standards in kDa. The original SDS-PAGE gels can be viewed from Additional file 9: Figures S6-S7. cZmDRIK1-KDR187-S514 binding assays for GTP and AMP-PNP as analyzed by ITC