The multifaceted allosteric regulation of Aurora kinase A

Abstract

The protein kinase Aurora A (AurA) is essential for the formation of bipolar mitotic spindles in all eukaryotic organisms. During spindle assembly, AurA is activated through two different pathways operating at centrosomes and on spindle microtubules. Recent studies have revealed that these pathways operate quite differently at the molecular level, activating AurA through multifaceted changes to the structure and dynamics of the kinase domain. These advances provide an intimate atomic-level view of the finely tuned regulatory control operating in protein kinases, revealing mechanisms of allosteric cooperativity that provide graded levels of regulatory control, and a previously unanticipated mechanism for kinase activation by phosphorylation on the activation loop. Here, I review these advances in our understanding of AurA function, and discuss their implications for the use of allosteric small molecule inhibitors to address recently discovered roles of AurA in neuroblastoma, prostate cancer and melanoma.

Keywords: allosteric regulation; aurora kinases; protein dynamics.

Figure 1.. AurA and the AGC-family kinases share a common regulatory architecture.

(A) Top: The domain architectures of AurA and Tpx2 are shown, highlighting the N-terminal domain and kinase domain of AurA, and the AurA-binding domain (AurA BD) of Tpx2. The kinase domain of AurA is divided into the N-lobe and C-lobe. The location of the A-box in the N-terminal domain of AurA, which targets the kinase for degradation, is also shown. Bottom: X-ray structure of the AurA kinase domain bound to residues 1–43 of human Tpx2 (PDB ID: 1OL5). The N-lobe and C-lobe are shown in beige and gray, respectively, the activation loop in blue and Tpx2 in magenta. The inset shows an expanded view of the docking site of Tpx2 on the N-lobe. (B) Top: A phylogenetic tree illustrating the relationship of the Aurora kinases to the AGC kinase family is shown on the left (adapted from ref. [24]), and the domain architecture of the AGC kinase PKA is shown on the right. Bottom: Structure of PKA (PDB ID: 1L3R) with the C-terminal tail containing the HM colored brown, the N-terminal extension colored yellow, and the remainder of the kinase domain colored as in (A). The inset shows the docking of the HM to the PIF pocket of the kinase.

Figure 2.. The unusual regulatory spine of AurA contains a functionally important water-mediated allosteric network.

(A) Left: Structure of the kinase domain core of PKA, colored as in Figure 1, with the four residues of the regulatory spine highlighted in dark gray. Right: Enlarged view of the regulatory spine of PKA, with the αC spine residue highlighted in yellow. (B) Enlarged view of the regulatory spine of AurA, colored as in (A), showing the interactions of the unusual Q185 αC spine residue with structured water molecules (red) co-ordinated to the DFG motif in the active site. (C) Histogram showing the frequency of different amino acid residues at the αC spine position across all ePKs. Values are derived from the original multiple sequence alignments published by Manning et al. [24]. (D) View of the water network in the AurA active site (PDB ID: 5G1X), showing the Q185 residue, three water molecules, the Glu-Lys salt bridge, and the bound ADP molecule. (E) Left: View of the active site of COT kinase showing the similar interactions of the glutamine αC spine residue with the active site water molecules (PDB ID: 4Y85). Right: View of the active site of PLK1 showing the water network mediated by the histidine αC spine residue (PDB ID: 2OWB). (F) Structure of SAR-156497 bound to AurA showing the interactions of the small molecule with the Q185 residue (PDB ID: 4UZD).

Figure 3.. Tpx2 binding to AurA triggers a conformational transition from a DFG-Out state to the active DFG-In state.

(A) Selected crystal structures of AurA in which the kinase adopts DFG-Out states with widely differing positioning of the activation loop [PDB IDs: 3UOK (yellow), 2WTV (brown), 5EW9 (beige) and 5L8K (dark purple)]. The active DFG-In state is highlighted for comparison (pink, PDB ID: 1OL5). Structures were aligned on the entire kinase domain. (B) Schematics of the dye-labeling scheme used to track the position of the activation loop by FRET (top), and (bottom) the distribution of inter-dye distances determined by time-resolved FRET for inactive AurA (dashed line), AurA activated by Tpx2 (pink), and AurA activated by phosphorylation on T288 (dark purple-shaded area). The data are taken from Ruff et al. [88]. (C) Structure of the probable DFG-Out state adopted by AurA in solution (purple). The flipped DFG motif is indicated, and the partially intact regulatory spine in which the W277 residue has taken the place of the DFG phenylalanine residue, is shown as a transparent surface. The conformation of the activation loop in the active DFG-In state is shown for reference (pink). The Cα atom of the residue labeled with a fluorescent dye is shown as a sphere in both structures. (D) Top: View of the active site in the active DFG-In state showing the bound nucleotide and magnesium ions, the DFG motif and the Asn 261 residue, which, together with the DFG Asp, co-ordinates one of the magnesium ions (PDB ID: 1OL5). Bottom: View of the active site in the crystal structure of the native DFG-Out state showing bound nucleotide, the flipped DFG motif and the Asn261 residue (PDB ID 5L8K). Magnesium coordination is lost in this state. (E) Illustrative free energy landscape for the DFG-In/DFG-Out transition, showing how nucleotide and Tpx2 binding lead to graded changes in the populations of the DFG-In and DFG-Out states. (F) Schematic of the AurA active site showing the DFG motif (light pink), αC-helix and Q185 residue (dark gray), water network (red spheres and yellow hydrogen bonds) along with bound Tpx2 (magenta) and ATP (light gray). The colored arrows represent the allosteric coupling of Tpx2 to the DFG motif through the water network (magenta), and the coupling of ATP to the DFG motif through the water network and magnesium ions (gray). The faded DFG-Out state (purple) represents the promotion of the DFG-In state by nucleotide and Tpx2.

Figure 4.. Phosphorylation of T288 triggers a rearrangement of the activation loop within the DFG-In state.

(A) Schematic of the spin-probe labeling scheme (top) and measured spin-spin distances (bottom) from double electron–electron resonance experiments on phosphorylated AurA (darker solid line and shading) and unphosphorylated AurA (lighter dashed line). Data are taken from Ruff et al. [88]. The inhibitor SNS-314 was used to isolate the DFG-In state. (B) Conformation of the activation loop in the active DFG-In substate promoted by phosphorylation on T288 (top, PDB ID: 1OL5), and putative structure of the autoinhibited DFG-In substate adopted in the absence of phosphorylation, identified in molecular dynamics simulations (bottom). The Cα atom of the residue on the activation loop labeled with the spin probe is shown as a sphere in both panels. (C) Schematics of the four main activation states of AurA highlighting the complementary structural and dynamic changes triggered by Tpx2 binding and phosphorylation (pT288), and the dynamically quenched state that results when these factors act together, e.g. in PP6-mutated melanoma cells.

Figure 5.. Defective anchoring of the AurA activation loop may underlie its heightened dynamics.

(A) Schematic representation of the activation loop of an active protein kinase, showing the DFG motif at the N-terminus, the two anchor points that stabilize the N- and C-terminal halves of the loop, and the hydrophobic latch residue at the end of Anchor point 1. The sidechains of several residues located on the anchor points and the underlying catalytic loop are shown, which form part of the interaction surface (dashed line) for the hydrophobic latch residue. (B) Sequence alignment of the activation loops of AurA and members of eight AGC kinase subfamilies. The alignment was manually curated based on X-ray structures of each kinase [PDB IDs: 4DC2 (PKCι), 4CRS (PKN2), 4EKK (AKT1), 4NW6 (RSK2), 1L3R (PKA), 4RQK (PDK1), 3NYN (GRK6), 4W7P (ROCK1), 1OL5 (AurA)]. In the lower inset, a comparison is shown of the structures of Anchor point 1 in PKCι (a kinase with a glycine insert before the hydrophobic latch residue) and PDK1 (a kinase lacking the insert), highlighting the similar positioning of the leucine hydrophobic latch residue in the two cases. (C) Crystal structures are shown highlighting the anchoring of the activation loop in AurA (blue) and the AGC kinases GRK6 (yellow) and PDK1 (beige). Atoms within 5.5 Å of the sidechain of the hydrophobic latch residue are shown in a surface representation (for clarity surfaces were excluded for the residues preceding and following the latch residue).

Figure 6.. Targeting AurA with allosteric small molecules.

(A) The structure of AurA bound to Tpx2 is shown on the right with the kinase domain in gray, Tpx2 shown in magenta, and the Tpx2-contact surface on AurA shown in pink. The top left panel shows an enlarged view of the structure of AurA bound to the allosteric small molecule AurkinA, showing AurkinA (blue) residing in the PIF pocket of the kinase. The same structure is shown underneath, aligned to the structure of free AurA in the DFG-In state (gray, PDB ID: 1OL7). Only the N-lobe is shown. (B) The locations of a representative subset of the small molecules found to bind to the Tpx2 binding surface by McIntyre et al. [98]. The molecules localize to three discrete pockets identified as the Y-, F-, and W-pockets based on the identity of the hydrophobic Tpx2 residues that occupy them in the AurA–Tpx2 complex. (C) Left: Structure of AurA bound to an N-terminal segment of N-Myc (residues 28–89). AurA is shown in gray with the activation loop highlighted in pink, and N-Myc is shown in yellow (PDB ID: 5G1X). The domain architecture of N-Myc is shown on the top right. The AurA-binding domain (AurA BD) is thought to overlap with the site of recognition by the ubiquitin ligase SCF^FbxW7. Right: Comparison of the conformation of the AurA activation loop in structures of the kinase bound to N-Myc (pink) and the DFG-Out kinase inhibitors MLN-8054 (purple, PDB ID: 2WTV) and CD532 (blue, PDB ID: 4J8M). Structures were aligned on the entire kinase domain.