Computational Studies Of Phosphorylation Induced Protein Conformation Changes

The reversible phosphorylation of proteins regulates almost all aspects of cell life, and provides a fast and efficient way to regulate protein functions, and it is used to control all basic cellular processes, including metabolism, signaling, motility, growth, proliferation, differentiation, organelle trafficking and membrane transport. Abnormal phosphorylation is a cause or consequence of many diseases. In many cases, phosphorylation regulates conformation changes of proteins and results in switch-like changes in protein function.From the nineties of the 20th century, with the development of computer science as well as the human genome project implementation, bioinformatics has been developed rapidly. Protein is an indispensable research object in bioinformatics, therefore, computer simulation of proteins has become a well-established and important research area. Via molecular modeling can build the three-dimensional model of proteins, to research the structure characteristics of the macromolecules, analyze the interaction between the protein and substrate, and describe protein biochemistry functions, thus molecular simulation has already become a powerful tool for biology experiments and drug design. Besides the conventional molecular dynamics simulation, the steered molecular dynamics simulation method became a good complement to it through modifying the energy function to effectively reduce the height of barriers separating low-energy states. In this thesis, we have carried out molecular simulation technology combined with steered molecular dynamics simulation and free energy calculations to study protein conformation changes and protein-inhibitor interactions induced by phosphorylation. The computer simulations of the protein and substrate interactions may understand some problems that can not been solved in the experiment. The simulation results can support the further studies on them and direct the design of new inhibitors and drugs. The main results are summarized as follows:1. Mechanism of Ser88 phosphorylation induced dimer dissociation in dynein light chain LC8.Dynein light chain LC8 is a highly conserved, dimeric protein involving in a variety of essential cellular events. Phosphorylation at Ser88 was found to promote mammalian cell survival and regulate the dimer to monomer transition at physiological pH condition. Combining molecular dynamics (MD) simulation and free energy calculation methods, we explored the atomistic mechanism of the phosphorylation induced dimer dissociation. The MD simulation revealed that phosphorylation/phosphomimetic mutation at Ser88 opens an entrance into the dimer interface for water molecules, which disturbs the hydrogen bond network around His55 and is expected to raise the pKa value and protonation ratio of His55 as well. The free energy calculations showed that S88E mutation destabilized the dimer by 6.6 kcal/mol, in good agreement with the experimental value of 8.1 kcal/mol. The calculated destabilization upon phosphorylation is 50.8 kcal/mol, showing that phosphorylation definitely prevents dimer formation at physiological condition. Further analysis of the calculated free energy changes demonstrated that the electrostatic contribution dominates the impact of phosphorylation on dimer dissociation.2. Mechanism of phosphorylation regulated interaction between ZO-1 PDZ2 and Cx43.The interactions of connexins (especially connexin43, Cx43), the most abundant connexin in mammals) with the second PDZ domain of ZO-1 received particular attention in recent years in the context of GJ formation and regulation. Accumulating evidence indicates that ZO-1 actively participates in the dynamic remodeling of GJs in a number of cellular systems, including cardiomyocytes, fibroblasts and neurons. There are evidences that the phosphorylation of connexin on the carboxyl terminal domain may regulate, or are correlated, with gap junction turnover, but the regulating mechanism is not clear yet. With molecular dynamics simulation, we explore the atomics mechanism of S(-9)/S(-10) phosphorylation regulated Cx43 binding with ZO-1 PDZ2 domain. The MD simulation shows that two monomers of PDZ2 domain have good flexibility and fluctuates toward each other during simulation whether binding with Cx43 or not. Cx43 with 12 residues shows better binding affinity than 9 residues peptide with PDZ2, while the latter forms 'hair-pin' during simulations. While S(-9) or S(-9,-10) was phosphorylated, the interaction between Cx43 peptide and PDZ2 was interrupted or weakened. Because the phosphate carries a -2 charge and the resulting large electrostatic perturbation modulates the energy landscapes governing protein-protein interaction. Hydrogen bonds formed by S(-9), S(-10) from Cx43 and E210, E238 from PDZ2 were disrupted because of phosphorylation. Long-Form Cx43 (with 12 residues) has the best binding affinity with PDZ2, in good agreement with experiment data.