| Cdc25B is a dual-specificity protein tyrosine phosphatase that catalyzes the dephosphorylation of the Cdk2/CycA protein complex. The Cdc25 phosphatases are important regulators of the human cell cycle, and have been identified as potential anticancer targets. However, drug discovery targeting Cdc25s has been hindered by a lack of experimental data regarding the nature of their interaction with their native protein substrates. In an effort to understand binding of Cdc25B with its natural protein substrate, we use rigid-body docking and molecular dynamics refinement to predict the docked orientation for Cdc25B with the Cdk2-pTpY-CycA protein substrate. The stable ensemble structure is validated by comparison with interaction free energies derived from double mutant cycle and hot-spot swapping experiments. Protein tyrosine phosphatases are thought to bind phosphate dianions and employ acid catalysis via the Asp residue in the highly conserved WPD-loop. However, Cdc25 phosphatases lack this motif present in many other phosphatases. We use quantum mechanical/molecular mechanical minimum free energy path calculations to determine the dephosphorylation mechanism of Cdc25B with a commonly used small molecule substrate. Specifically, we simulate the rate-limiting step of the reaction catalyzed by Cdc25B with the substrate p-nitrophenyl phosphate in both the monoanionic and dianionic forms of the substrate. Surprisingly, our calculations favor a mechanism involving the monoanionic form. Thus, Cdc25 may employ a unique dephosphorylation mechanism among protein tyrosine phosphatases, at least in the case of the small molecule substrate p-nitrophenyl phosphate.;Additionally, we use quantum mechanical/molecular mechanical calculations to understand the interactions that govern ligand binding in hepatitis C virus polymerase NS5B. Our calculations show that the most important energetic component is the ligand internal energy. We then describe the quantum mechanical/molecular mechanical minimum free energy path method for determining accurate reaction paths and energetics for enzymatic and solution-phase reactions. We test the method on the second proton transfer step of the reaction of 4-oxalocrotonate tautomerase with the substrate 2-oxo-4-hexenedioate. Finally, we provide new parameters for the pseudobond method for use in quantum mechanical/molecular mechanical simulations. Parameters for Cps (sp 3)--Cps(sp3), C ps(sp3)--Cps(sp 2,carbonyl) and Cps(sp3)N(sp 3) pseudobonds are obtained and provide significantly improved electrostatics relative to previous pseudobond parameterizations. |
