Fig 1.
Architecture of the RTKC8 receptor tyrosine kinase and purification of its kinase domain.
Domain organization of full length RTKC8, consisting of Cys-rich and hyalin rich (HYR) domains in the extracellular domain, followed by a transmembrane domain (TM) and an intracellular domain. The intracellular domain contains a juxtamembrane (JM) domain, a kinase domain, and a C-terminal tail with tyrosine residues (red circles) and a CAP-Gly domain.
Fig 2.
Phosphorylated RTKC8 kinase domain is active and phosphorylates tyrosine residues in tail segment peptides.
(A) The RTKC8 kinase domain was dephosphorylated by YopH phosphatase and samples were taken at different time intervals and probed with an anti-phosphotyrosine antibody. The control showed that the band intensity on the western blot for the kinase domain without YopH is unchanged over 2 hours at room temperature while the band intensity decreased rapidly upon the addition of the tyrosine phosphatase YopH. (B) The sequence of tyrosine phosphosite peptides (PKCδ-Y313, PDGFRα-Y762, RTKC8-Y1828, RTKC8-Y1897, RTKC8-Y1990) used as substrates for the kinase (C) Phosphorylation activity of RTKC8 kinase domain on the tyrosine containing peptides PKCδ-Y313 and PDGFRα-Y762 (D) Phosphorylation activity of RTKC8 kinase on tyrosine residue containing peptides from the tail segment (RTKC8-Y1828, RTKC8-Y1897, RTKC8-Y1990). Kinase assays were performed in duplicate.
Fig 3.
The RTKC8 kinase domain crystallized in an active conformation with staurosporine.
(A) The RTKC8 structure reveals a classic, two lobed kinase domain (teal) with an αC helix (blue), P-loop (green), activation loop (red), catalytic loop (violet) with two staurosporines bound, one at the active site and the other at the peptide-substrate binding site (light orange). Two views are shown. The one on the right is rotated slightly from the left view so as to show the break in the activation loop more clearly. (B) The active αC-helix-in conformation is demonstrated by the presence of a salt bridge between the conserved αC-helix Glu1592 and Lys1575 in the β3 strand (distance between the two Cβs is <10Å; distance between the Lys ε-amnino and the Glu carboxyl is 3.4Å), the presence of a hydrogen bond between the backbone of the amino acid immediately prior to the DFG and the His in the HRD motif (3.1 Å) and an extended activation loop with a hydrogen bond between backbone carbonyl group of Arg1689 of the DFGxxR motif and the backbone amino group of Ile1663 which is immediately before the HRD motif. A hydrogen bond in the extended activation loop between the DFG motif Phe1685 backbone carbonyl and the amino nitrogen of Ala1688 (DFGxA, 3.2 Å) presents another feature of an active conformation of a kinase. (C) The second staurospaurine binds at the peptide-substrate binding site. When the structure of the human insulin kinase domain (yellow) in complex with a peptide substrate (1IR3) is overlayed on the RTKC8 kinase domain (cyan), the second staurospaurine (light orange) can be seen overlapping the position of the C-terminal segment of the substrate peptide (magenta) bound between the activation loop, the αEF helix and the αG helix.
Fig 4.
Two molecules of staurosporine (STU1 and STU2) are bound to the RTKC8 kinase.
(A) One molecule of staurosporine (STU1) is bound at the active site, surrounded by the N-lobe β strands, the activation loop and the small helix between αE and β6 of the C-lobe (electron density for the Fo-Fc map with no compound modeled contoured at 2.0σ in grey). (B) The other staurosporine (STU2) is bound to the C-lobe, next to the activation loop, the αEF helix and the αG helix (electron density for the Fo-Fc map with no compound modeled contoured at 2.0σ in grey). (C) The contacts of STU1 in the active site of the RTKC8 kinase domain with side chain residues. Dotted lines (green) represent the hydrogen bonds and arches (red) represent the hydrophobic contacts (prepared by LigPlot+ [38]) (D) Contacts for the other saturosporine (STU2) prepared by LigPlot+.
Table 1.
Structure determination and refinement of RTKC8 kinase domain bound to staurosporine.
Fig 5.
Structure and sequence alignment of RTKC8 kinase and EphA5 kinase.
A Dali search identified the kinase domain of EphA5 (2R2P) as the structure with the highest similarity to RTKC8 in the PDB. The RTKC8 kinase (cyan) and EphA5 kinase (yellow) structures were aligned with a rmsd 0.836 Å. B. Sequence alignment of the EphA5 kinase domain with the RTKC8 kinase domain, with the secondary structural elements above the alignment. The alignment prepared with ESPript 3.0 [44].