PMC

Crystals of TTBK1 obtained in the presence of (a) staurosporine and (b) compound 3.

Conversion of 3-bromo-5-methoxyaniline to compound 3. a, 3-Bromo-5-methoxy­aniline, acetonitrile, heat and then 4-methylpiperidin-4-amine, DIPEA, microwave reaction; b, BBr₃, dichloromethane.

SDS–PAGE analysis of (a) TTBK1(14–343) and (b) TTBK1(14–320) purified from baculovirus. The samples were electrophoresed on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and stained with SimplyBlue Coomassie stain (Invitrogen). Lane 1, molecular-mass markers (kDa); lane 2, 1 µg protein; lane 3, 3 µg protein; lane 4, 6 µg protein. The arrow to the right of lane 4 in (a) indicates the proteolytic fragment.

(a) Schematic diagram of human TTBK1 (as derived from Sato et al., 2006). TTBK1 includes a dual kinase domain (KD), a conserved domain A (DA), a polyglutamate domain (PG) and a conserved domain B (DB). (b) The amino-acid sequence of the TTBK1 constructs that were designed for crystallization studies showing the longer construct (residues 14–343) and the shorter construct (residues 14–320). The N-terminal His₆ affinity tag and TVMV protease cleavage site are underlined. Non-native residues following TVMV cleavage are denoted as red letters. Residues encompassing the TTBK1 kinase domain are denoted as cyan letters.

Structure of apo TTBK1. (a) The final 2F _o − F _c electron-density map is shown with the final model of the activation loop (178–202), showing that the electron density is reasonably good throughout the region. (b) Cartoon diagram of the superposition of CK1 (PDB entry 1cki) on TTBK1, with TTBK1 shown in cyan and 1cki shown in red. The C-terminal domains of CK1 and TTBK1 were superimposed and the transformation matrix was then applied to the entire 1ck1 structure, showing that the N-terminal domain of TTBK1 has rotated relative to its position in the 1cki structure. (c) After the superposition described in (b) the N-terminal domain of 1cki was superimposed on the N-terminal domain of TTBK1. This required an additional 9° rotation about the axis shown in both (b) and (c). This shows that the N-terminal domains superimpose reasonably well except for some local variation, for example around residues 45 (the P-loop), 69 and 100 in TTBK1, which correspond to residues 22, 47 and 78, respectively, in 1ck1. (d) The active site is shown with water molecules 501, 502, 503 and 504 labelled. Hydrogen bonds are shown as a series of black prolate ellipsoids.

Binding of compound 3 to TTBK1. (a) Two-dimensional representation of compound 3 oriented similarly to that in (b). An IUPAC name for compound 3 is 3-({5-[(4-amino-4-methylpiperidin-1-yl)methyl]pyrrolo[2,1-f][1,2,4]triazin-4-yl}amino)-5-bromophenol. (b) Initial F _o − F _c electron density contoured at 3 r.m.s.d. (slate) and 10 r.m.s.d. (orange) with the final model of the TTBK1–compound 3 complex. The hinge region (residues 108–111) is at the top of the figure and the gatekeeper residue (Met107) is on the right. C atoms are shown in cyan for TTBK1 and in orange for compound 3, N atoms are shown in blue, O atoms are shown in red, S atoms are shown in yellow, the Br atom is shown in violet and waters are represented by red spheres. (c) Cartoon representation of the placement of compound 3 in the context of the entire TTBK1 kinase domain, with a superposition of the apo structure on the complex structure. The complex structure is shown in cyan, the apo structure is shown in magenta and compound 3 is shown with C atoms in orange, N atoms in blue, the O atom in red and the Br atom in violet. (d) Superposition of the apo structure at the binding site on the structure of the complex with compound 3. The hinge region (residues 108–111) is at the top of the figure. C atoms are shown in cyan for the complex structure and magenta for the apo structure, N atoms are shown in blue, O atoms are shown in red, S atoms are shown in yellow, the Br atom is shown in violet and water molecules are shown as red spheres. Hydrogen bonds are shown as a series of black prolate ellipsoids. The figures show that only the side chains of Glu77, Met107 (gatekeeper residue) and to a lesser extent Asp176 move significantly upon binding compound 3.