Structure of Aurora B-INCENP in complex with barasertib reveals a potential transinhibitory mechanism

Abstract

The Aurora family is a well conserved and well characterized group of serine-threonine kinases involved in the normal progression of mitosis. The deregulation of Aurora kinases impairs spindle assembly, checkpoint function and cell division. To date, many small molecules that compete with ATP for binding to Aurora kinases have been developed and characterized. Here, the first structure of the Xenopus laevis Aurora B-INCENP complex bound to the clinically relevant small molecule barasertib was determined. The binding properties of this inhibitor to the Aurora B active site are analyzed and reported. An unexpected crystal-packing contact in the Aurora B-INCENP structure coordinated by an ATP analogue is also reported, in which the INCENP C-terminus occupies the substrate-binding region, resembling the protein kinase A inhibitory mechanism.

Keywords: Aurora kinases; PKA; barasertib; cancer; small-molecule inhibitors.

Figure 1

Binding of barasertib to the ATP-binding pocket of Aurora B. (a) Chemical structure of barasertib prodrug (red) and the active form (green). (b) The three-dimensional structure of Aurora B in cartoon representation, illustrating the N-terminal small lobe (grey), the C-terminal helical large lobe (white), the short C-terminal extension (green), the αC helix (blue) and the activation loop (red). INCENP (orange) crowns the small lobe of Aurora B, stabilizing an active conformation of the kinase. The active form of barasertib (sticks) and its unbiased F _o − F _c OMIT electron-density map (grey mesh) occupy the ATP-binding pocket at the interface between the small and large lobes. (c) Stick representation of the interaction between the active form of barasertib (green) and selected residues of Aurora B. (d) Structure superimposition of the Aurora B–INCENP–barasertib active form complex (grey) with the Aurora A–TPX-2 complex (cyan; PDB entry 1ol5; Bayliss et al., 2003 ▶). O, N and S atoms are shown in red, blue and yellow, respectively. Hydrogen bonds are shown as dashed lines and bond lengths are indicated in Å.

Figure 2

The INCENP C-terminal extension occupies the Aurora B substrate-recognition surface. (a) The arrangement of Aurora B–INCENP molecules in the asymmetric unit is shown in cartoon and ribbon format in grey (Aurora B) and orange (INCENP). The C-terminal portion of INCENP packs against the Aurora B large C-lobe. (b) Details of the INCENP IN-box (orange) molecular interactions with the Aurora B kinase domain (grey). Calculated electron-density maps for the AMP-PNP and for the INCENP segment are drawn in green and brown, respectively. O, N and S atoms are shown in red, blue and yellow, respectively. (c) Multiple sequence alignment of the INCENP C-­terminal region from different species. The alignment is colour-coded based on conservation. A solid red bar indicates the INCENP stretch occupying the Aurora B substrate-recognition surface. Numbering is according to the X. laevis sequence. (d, e) Surface representations of Aurora B with INCENP (d) and of protein kinase A with PKI (PDB entry 1atp; Zheng et al., 1993 ▶) (e). Surfaces are coloured according to the electrostatic potential distribution of the kinases, ramped from blue (positive) to red (negative). (f) Schematic representation of an Aurora B potential transinhibitory model proceeding through an intermolecular mechanism, which may be enhanced by a high local protein concentration. This transinhibition mechanism may be relieved by a pTSS–pThr248 repulsion and lead to full activity of Aurora B. A Ser/Thr phosphatase can then revert the full Aurora B activity to a transinhibited state.