Flexibility and inhibitor binding in cdc25 phosphatases

Abstract

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

Conformational flexibility of the complete catalytic domain of Cdc25B phosphatases

“…The global flexibility of Cdc25B has been debated in the literature . Previous simulations employing either molecular dynamics with the OPLS‐AA force field or a library of rotamers sampled by Monte Carlo suggested that the Cdc25B C‐terminal α ‐helix may (partially) unfold in solution.…”

“…The global flexibility of Cdc25B has been debated in the literature . Previous simulations employing either molecular dynamics with the OPLS‐AA force field or a library of rotamers sampled by Monte Carlo suggested that the Cdc25B C‐terminal α ‐helix may (partially) unfold in solution. NMR experiments presented previously for a truncated construct of the Cdc25B catalytic domain and obtained here for the complete catalytic domain show this C‐terminal α ‐helix is folded and has little flexibility.…”

“…Previous computer simulations suggested that the C‐terminal α ‐helix observed in Cdc25B could be (partially) unfolded in solution . But, recent nuclear magnetic resonance (NMR) experiments refuted this conclusion and provided evidence that the C‐terminal helix has little flexibility and is well folded in the ps–ms time scale .…”

“…Although the homologous Cdc25C has a 100‐fold lower activity toward Cdk2‐pYpT/Cyclin‐A, a chimeric construct with the last 18 residues from the Cdc25B C‐terminal tail fused to the Cdc25C catalytic core shows a gain‐of‐function with a 50‐fold increase in activity . Given the spatial proximity of the C‐terminus and the P‐loop in the Cdc25B structure, the terminal region could play important roles in complexation processes such as: release and egress of the product inorganic phosphate, binding and stability of bulky artificial phosphatase substrates such as O‐methyl fluorescein phosphate, and complexation of competitive inhibitors …”

Modeling loop backbone flexibility in receptor‐ligand docking simulations

“…[34] Furthermore, backbone flexibility was applied to different therapeutically important targets. [36–38] For a recent review see Refs 39, 40…”