Structural mechanism of synergistic activation of Aurora kinase B/C by phosphorylated INCENP

Abstract

Aurora kinases B and C (AURKB/AURKC) are activated by binding to the C-terminal domain of INCENP. Full activation requires phosphorylation of two serine residues of INCENP that are conserved through evolution, although the mechanism of this activation has not been explained. Here we present crystal structures of the fully active complex of AURKC bound to INCENP, consisting of phosphorylated, activated, AURKC and INCENP phosphorylated on its TSS motif, revealing the structural and biochemical mechanism of synergistic activation of AURKC:INCENP. The structures show that TSS motif phosphorylation stabilises the kinase activation loop of AURKC. The TSS motif phosphorylations alter the substrate-binding surface consistent with a mechanism of altered kinase substrate selectivity and stabilisation of the protein complex against unfolding. We also analyse the binding of the most specific available AURKB inhibitor, BRD-7880, and demonstrate that the well-known Aurora kinase inhibitor VX-680 disrupts binding of the phosphorylated INCENP TSS motif.

Fig. 1

Phosphorylated INCENP binds the activation loop of Aurora kinase B/C. a Structure of activated AURKC bound to the phosphorylated INCENP “IN-box”. b, c Views from two angles showing how the phosphorylated INCENP makes extensive hydrogen-bonding interactions with the activation loop and αC-helix of AURKC, and contributes significantly to arranging the active conformation and substrate binding region of the kinase. d Sequence alignment of the αC and activation loop regions of AURKB or AURKC from diverse organisms. Residues involved in binding INCENP are marked with triangles below the alignment. For a full sequence alignment and sequence accession numbers see Supplementary Fig. 4. The sequence alignment was created using ClustalO and Aline. e In the fully active AURKC:INCENP structure INCENP makes contact with the Aurora kinase N-lobe and C-lobe, and the activation loop. f, g Views from two angles showing how INCENP binds on two sides of AURKC αC including an aromatic stacking interaction between INCENP W897 and AURKC H97 and H190, as well as the hydrogen-bonding network around INCENP pS893 and pS894. These binding interactions link AURKC αC and activation loop into a stable conformation, and the INCENP phosphorylations help to form the substrate binding groove, thus activating AURKC to different substrates

Fig. 2

Enzymatic assays of AURKB or AURKC with wild-type or mutant INCENP. Mutation of the INCENP phosphorylation sites Ser893 or Ser894, or of INCENP Trp897 which is also important for Aurora kinase:INCENP binding, altered the enzymatic properties considerably. All measurements were made in triplicate. a Loss of TSS motif binding decreases the reaction rate for both AURKB and AURKC on a peptide substrate. b Quantification of the rate of reaction during the linear reaction phase of a. Error bars show standard error from the linear regression. c Measurement of initial rate of reaction (V₀) at varying peptide substrate concentrations, with curves calculated by non-linear regression (curve-fitting) to the Michaelis–Menten equation. Calculated K_M and K_cat values from non-linear regression are shown in Table 2; the mutants show significant variation in K_M but similar K_cat values (with the possible exception of S894A)

Fig. 3

TSS motif binding affects AURKC:INCENP binding and shape. a, b Models of the effect of loss of TSS motif binding from a phosphorylated and ordered INCENP TSS motif as seen in the crystal structure (a) to an unphosphorylated TSS motif and disordered C-terminal part of INCENP (b). c Native mass spectra of AURKC:INCENP complexes show that under the same gentle ionisation conditions the wild-type AURKC:INCENP complex remains intact while the non-phosphorylated INCENP mutants are only partially bound. This is shown by the single peak series around m/z 3000–3200 for the wild-type AURKC:INCENP complex (MW expected 40501 Da, observed 40747 Da; one AURKC phosphorylation and two INCENP phosphorylations), while there are separate charge state series for the individual AURKC and INCENP proteins when samples of INCENP mutant complexes were measured (AURKC:INCENP-S894A MW expected 40059 Da, observed 40224 Da; one AURKC phosphorylation and one INCENP phosphorylation, and AURKC:INCENP-S893A/S894A/W897A MW expected 39928 Da, observed 40009 Da; one AURKC phosphorylation). d, e Without a phosphorylated INCENP TSS motif the AURKC:INCENP complexes have shorter ion mobility drift times (12+ and 13+ charge states shown). f Collision-induced unfolding shows the AURKC:INCENP mutant complexes reach the greatest extension already at lower energies consistent with greater structural flexibility. g The experimental CCS values of the AURKC:INCENP complexes were calculated after calibration from the ion mobility drift times. At all charge states the INCENP mutant complexes have a reduced collision cross-section consistent with a reduced size of the compact, ordered and folded protein domain

Fig. 4

VX-680 binding partially disrupts AURKC:INCENP and affects the substrate binding groove. a Crystal structure of AURKC:INCENP:VX-680. AURKC is in magenta for the N-lobe, blue for the C-lobe and orange for the activation loop. INCENP is shown in green, VX-680 in yellow. The parts of the AURKC:INCENP:BRD-7880 structure that differ in position from the structure with VX-680 are shown in grey, illustrating the parts of AURKC:INCENP disrupted by VX-680 binding. b VX-680 binding to AURKC disrupts INCENP binding to the activation loop, disrupting the substrate binding surface. INCENP is shown in green and the part of INCENP not observed in the crystal structure is shown in grey, based on the structure of AURKC:INCENP:BRD-7880. c Binding of INCENP Trp897 to the C-terminal part of AURKC αC is maintained in the presence of VX-680. d A crystal structure of AURKA with BRD-7880 shows that the binding mode of BRD-7880 is conserved. Comparison of this structure with previously published structures of AURKA with VX-680 shows that, as with AURKC, BRD-7880 allows an Aurora kinase conformation resembling the active form exemplified by AURKA:TPX2:ADP, while VX-680 causes a rotation of the N-lobe that disrupts the positions of αB and αC. A 2F_o–F_c electron density map is shown in blue around BRD-7880, contoured at 1.0 σ (0.14 eÅ⁻³)

Fig. 5

BRD-7880 binding to AURKC:INCENP. a BRD-7880 forms a single hydrogen bond to the kinase hinge region from its benzo−1,3-dioxole moiety, and multiple hydrogen bonds to the P-loop (Lys53) and to Lys72 and Glu91. A 2F_o-F_c electron density map is shown in green around BRD-7880, contoured at 1.0 σ (0.4 eÅ^-3) b The central 8-membered ring of BRD-7880 allows excellent shape complementarity to the ATP-binding site of AURKC. c Isothermal titration calorimetry confirmed BRD-7880 has significantly higher affinity for AURKB:INCENP over AURKA, due to more favourable entropy of binding (Supplementary Table 2). d Selectivity of BRD-7880 for AURKB:INCENP over AURKA:TPX2 is increased due to a non-ideal interaction, most likely the forced, possibly partial, dissociation of TPX2 from AURKA upon BRD-7880 binding. e Chemical structure of BRD-7880. f BRD-7880 has slow binding kinetics with AURKB:INCENP, with the long half-life of dissociation contributing to its potent binding. g BRD-7880 binds to AURKC:INCENP and AURKA in the same conformation, with the active site residues of AURKA adopting the same conformations as those of AURKC, with the exception of the DFG motif (residues 274 to 276 of AURKA). AURKC is shown in blue with bound BRD-7880 in yellow, and AURKA is shown in green