Structural comparison of human mammalian ste20-like kinases

Abstract

Background: The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains.

Principal findings: We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family.

Significance: The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design.

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Figure 1. Alignment of human MST kinase domains.

The secondary elements shown in the alignment were derived from the structure of MST4 (PDB ID 3GGF). The conserved Asp residue position that is involved in the coordination of ATP via its ribose oxygen atoms is marked with a yellow arrow.

Figure 2. Overall structure of the MST kinase domains.

Superimposition of the C-lobe from the three MST structures: Active apo MST1 (magenta, PDB ID: 3COM); Active MST3 (green, PDB ID: 3A7J) in complex with ADP (sticks) and Mn²⁺ ions (spheres); Inactive MST4 (blue, PDB ID: 3GGF) in complex with a quinazoline inhibitor (sticks) and Cd²⁺ (sphere). Phosphorylated threonine residues (Tpo) are shown as sticks.

Figure 3. ATP binding site.

Different kinases are color coded as follows: MST1: magenta; MST3: green; MST4: blue. Binding site surfaces are represented by semi-transparent orange skins. A. MST1 apo (PDB ID: 3COM); B. MST3 in complex with staurosporine (PDB ID: 3CKX); C. MST3 in complex with ADP (PDB ID: 3A7J); D. MST4 in complex with a quinazoline inhibitor (PDB ID: 3GGF); E. Fo-Fc difference map of the quinazoline inhibitor bound to MST4 (PDB ID: 3GGF). Isosurface contouring levels: 1σ (blue) and 3σ (red). This view is rotated 90° on the X-axis (as compared to the orientation shown in (D) and looks towards the C-lobe; F. Details of the active site of MST4 in complex with a quinazoline inhibitor (blue), superimposed to PAK6 (yellow; PDB ID: 2C30) and TAO2 in complex with ATP (grey; PDB ID: 1U5R).

Figure 4. Selected hits from screen of potential MST4 inhibitors against a panel of 1457 compounds.

Figure 5. Positioning of αC helix in the structure of three MSTs.

The kinase domains were superimposed at the N-lobe. MST1 (magenta), MST3 (green) and MST4 (blue). PAK6 (yellow wireframe) has been added to the superimposition to highlight the position typically occupied by the αC helix, in an active kinase.

Figure 6. MST4 dimer seen in the asymmetric unit.

Chain A is shown in blue, chain B is shown in orange. A. Overall packing. Cadmium ions are shown as yellow spheres, ligands are shown as sticks; B. Molecular interactions seen in the A-loop. Residue Thr178 (to be phosphorylated) is shown with red labels; C. Surface electrostatics potential in the dimerization region – the segment involved in the interaction from the chain B is shown in sticks representation.

Figure 7. Self association and autophosphorylation of MST4 in solution.

A: Self association studied by analytical ultracentrifugation. Shown are sedimentation velocity data. The sedimentation coefficients have been normalized to standard condition. Peaks corresponding to the MST4 monomer and dimer are indicated by “M and D” respectively. B: Autophosphorylation of MST4 studied by ESI-MS. The mass of the unmodified protein (33.818 Da) has been indicated. In addition to the three detected phosphorylation events the protein also oxidized (+16 mass shifts indicated by *).

Figure 8. αK and αD conformations for the two subfamilies of MSTs.

A. MST1 (magenta) adopts a conformation closer to that seen in TAO2 (red); B. MST3 and 4 (green and blue, respectively) adopts a similar fold to that of PAKs (yellow).

Figure 9. Surface electrostatic potential of MSTs.

Crystallographic models are labeled in blue; homology models are labeled in orange. The surface electrostatic potential is represented as a color ramp, ranging from −5 kcal/e.u. charge (red) to blue +5 kcal/e.u. charge (blue).