Volume 18, Issue 3, Pages 379-391 (April 2005) Mechanism of Aurora B Activation by INCENP and Inhibition by Hesperadin  Fabio Sessa, Marina Mapelli, Claudio Ciferri, Cataldo Tarricone, Liliana B. Areces, Thomas R. Schneider, P. Todd Stukenberg, Andrea Musacchio  Molecular Cell  Volume 18, Issue 3, Pages 379-391 (April 2005) DOI: 10.1016/j.molcel.2005.03.031 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of the Aurora B:IN-box Complex (A) Scheme of X. laevis Aurora B and INCENP. (B) SEC elution profile of Aurora B:INCENP790–847 complex. Eight 50 μl fractions between 1.5 and 1.9 ml were analyzed by SDS-PAGE. (C) Phosphorylation of the Aurora B:INCENP complex. Top: Coomassie-stained SDS-PAGE gel of Aurora B and Aurora B:INCENP samples. Middle: Western blotting with an antibody against phospho-Thr248. Aurora B (lanes 1 and 2) is phosphorylated on Thr248 even in the absence of INCENP. Treatment with PP1 removes phosphorylation on Thr248. Bottom: Mobility of INCENP790–856 (lanes 5 and 6) modified by PP1 treatment. (D) Aurora B samples were incubated in a kinase assay with histone H3. Top: loading control. Bottom: Aurora B:INCENP790–856 complex consistently showed an ∼7-fold activation relative to the Aurora B:INCENP790–847 complex. Molecular Cell 2005 18, 379-391DOI: (10.1016/j.molcel.2005.03.031) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Overall View of the Aurora B:IN-box Complex (A–C) Three views related by 90° rotations of the Aurora B:INCENP790–847 complex (gray and orange, respectively) also showing activation segment (red), phosphorylated Thr248 (yellow), C-terminal tail (green), and αC helix (blue). INCENP forms a crown around the N-lobe. Ribbon diagrams were created with PyMol (http://www.pymol.org). (D) Alignment of Aurora and INCENP sequences with secondary structure. The alignment is color coded based on conservation measured on a larger alignment of seven Aurora B and eight INCENP sequences. Orange dots mark Aurora B residues contacting INCENP. Molecular Cell 2005 18, 379-391DOI: (10.1016/j.molcel.2005.03.031) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Details of the Aurora B:IN-box Interaction (A) The Aurora B:INCENP complex is viewed as in Figure 2C. (B–F) Close-ups of the interaction of INCENP790–847 with Aurora B:INCENP complex is shown from N to C terminus in subsequent snapshots as indicated in (A). Only key contacts are shown. Molecular Cell 2005 18, 379-391DOI: (10.1016/j.molcel.2005.03.031) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of the Aurora B:INCENP Complex and the Aurora A:TPX2 Complex (A) Left: surface of Aurora A (gray) with ribbon model of TPX2 (red). The dashed arrow represents the direction of the TPX2 chain. Right: ribbon model of the Aurora A:TPX2 complex in the same orientation. (B) Aurora B:INCENP complex with same orientation and style as (A). (C) The main chains of TPX2 (red) and INCENP (orange) are shown on the surface of Aurora B after superposition of the Aurora N-lobes. Tyr825-Phe837 and Tyr8-Phe19 (aromatic plug) occupy similar positions. (D) GST-Aurora B was coexpressed with INCENP790–847 or the indicated mutants. Coomassie-stained gels show affinity-purified GST-Aurora B (top) and copurified INCENP (bottom). Molecular Cell 2005 18, 379-391DOI: (10.1016/j.molcel.2005.03.031) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Activation Mechanism of Aurora B (A) The activation loop of the Aurora B:INCENP complex is phosphorylated on Thr248 and identical to that of the Aurora A:TPX2 complex, which is doubly phosphorylated on Thr287 and Thr288. (B) Superposition with the activation loop of apo-Aurora A shows poor fitting. (C) Superposition of the N-lobes of the Aurora B:INCENP complex and the Aurora A:TPX2 complex. K122Au-B and K162Au-A occupy similar positions. E141Au-B is rotated away from the position of E181Au-A, and its distance from K122Au-B is ∼5.4 Å. The rotation depends on F837INCENP pushing against L138. The new position of Glu141Au-B is incompatible with the small interlobar angle of the Aurora A:TPX2 complex, as it predicts a clash of Glu141 with the activation loop (dashed circle). (D) The catalytic cleft of the Aurora B:INCENP complex is ∼15° more open than in the Aurora A:TPX2 complex, shown in (E). The approximate position of a rotation axis describing the rotation is shown. The arrowheads in (D) and (E) indicate equivalent points in the two structures that reveal the rotation. (F) A model describing the state of activation of the Aurora B:INCENP complex in the absence of INCENP phosphorylation. The activation loop is fully stretched. F837 pushes on L138 to rotate the αC helix. The rotated E141 is unable to bind K122 and pushes on the activation loop, opening the cleft. The C-terminal extension of Aurora B stabilizes this conformation. (G) TSS phosphorylation releases the pressure of F837. αC rotates back into normal position, closing the cleft, releasing the C-terminal brake, and restoring the E141-K122 ion pair and full activation. (H) Kinase activity assays on Histone H3 (bottom) with different variants of Aurora B as described in the text. The first three panels are Coomassie-stained loading controls. Molecular Cell 2005 18, 379-391DOI: (10.1016/j.molcel.2005.03.031) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Binding of Hesperadin to the Aurora B:INCENP Complex (A) Hesperadin (yellow) is an active site inhibitor of Aurora B. (B) Electron density omit map of Hesperadin bound to Aurora B. (C) LIGPLOT (Wallace et al., 1995) view of the interactions of Hesperadin with Aurora B. Molecular Cell 2005 18, 379-391DOI: (10.1016/j.molcel.2005.03.031) Copyright © 2005 Elsevier Inc. Terms and Conditions