Molecular Modelling Study On Structure And Function Of Protein Kinase A And Its Inhibitor

Protein kinase A(PKA) is a Ser/Thr protein kinase, bearing an important role in signal transduction. The PKA holoenzyme(R₂C₂) consists of two regulatory(R) and two catalytic(C) subunits, which is dissociated in response to binding the second messenger, cyclic 3', 5'-adenosine monophosphate(cAMP). The catalytic subunit that serves as a model for the protein kinase family is research hotspot in the recent years. PKA has regulatory roles in cell proliferation, differentiation, gene expression and glycogen metabolism. PKA is the primary potential target for therapeutic agents, since its unregulated activity has been implicated in many diseases. The thesis is about the theoretical calculation on the functional role of phosphorylation and structural water molecules in catalytic subunit and the binding mode of catalytic subunit of PKA with its inhibitor.Phosphorylation of Thr-197 in the activation loop of the catalytic subunit of PKA is an essential step for its proper biological function. In order to explore the influences triggered by phosphorylation, 5 ns comparative molecular dynamics(MD) simulation studies were performed on the complex of C-subunit bound to ATP and two Mg²⁺ ions (C/Mg₂ATP complex) and on the complex of C-subunit bound to its substrate peptide(C/PKS complex). For each complex, two MD simulaton studies were performed, one with phosphate group on Thr-197 and Ser-338, the other without. The results present an increasing flexibility of the activation loop in the unphosphorylated states, however, the unphosphorylated activation loop does not block the substrate-binding site, which is in good agreement with experimental result that unphosphoylation does not decrease the binding rate for protein kinase substrate. The conformation of C-helix is important for activity of protein kinase. In unphosphorylated state, the crucial interaction between Thr-197 and His-87 of C-helix does not exist, the C-helix runs away from its original position, which eventually displaces the phosphates group of ATE Thus the phosphoryl transfer in-line manner is demolished, which reduces the rate in phosphoryl transfer.Water molecule plays an important role in structure, stability, dynamics and function of biomolecule. In order to study the influence triggered by the three water molecules buried within the large lobe of catalytic subunit of PKA, two 2.5 ns molecular dynamics(MD) simulation studies were performed on catalytic subunit bound to its inhibitor BD2(C: BD2 complex), one with the three water molecules, the other without. The results show that, the three water molecules not only reduce the flexibility of catalytic subunit by forming strong hydrogen bonds with their neighboring residues, but also influence the binding mode between inhibitor BD2 and catalytic subunit. Though the three water molecules were located in large lobe, they are in the network of interactions that extended outward from the active site cleft. In the simulation without the three water molecules, the lack of crucial interaction between Tyr-164 and Thr-183 causes the rearrangement of Thr-183 and Asp-184, which demolishes their interactions with BD2.Due to the remarkably high sequence identity and three dimensions conservation of ATP binding pocket among the protein kinase, the design of selective inhibitors for protein kinase becomes a difficult task in the field of medicine and signal transduction. In order to elucidate the mechanism of the high selectivity of BD2 for PKA over PKC, using the crystal structure of catalytic subunit complexed with BD2 as the template, model of PKCβⅡwas built by homology modeling and its complex with BD2 was constructed by molecular docking, and 2.5 ns molecular dynamics simulation studies were carried out to compare the binding mode of BD2 with PKA and PKCβⅡ. The result shows that BD2 forms seven hydrogen bonds with PKA, while it forms five hydrogen bonds with PKCβⅡ. Thr-183 of PKA forms hydrogen bond with BD2, however, Ala-483 that is aligned to Thr-183 of PKA can not offer the hydrogen bond acceptor and does not formed hydrogen bond with BD2. The hydrophobic interaction between BD2 and PKA is stronger than that between BD2 and PKCβⅡ, which is mainly contributed by the difference in the hydrophobic interaction of the A ring and B ring of BD2 with this two protein kinases. Thedifference between binding mode of BD2 with PKA and PKCβⅡexplains the high selectivity of BD2, which provides the basis for further designing and optimizing balanol analogue inhibitor. In order to seek the rule in designing and optimizing 3, 5-disubstituted pyridine inhibitor, molecular dynamics simulation study was performed on PKA and inhibitor (the template compound in CoMFA, No. 29) complex to explore the binding mode of the inhibitor and PKA. Then three dimension quantitative structure-activity relationship(3D-QSAR) studied were formed by CoMFA and CoMSIA. The result of molecular dynamics simulation study shows that the nitrogen of the terminal pyridine interacts to Val-123 via a hydrogen bond, the nitrogen of the central pyridine hydrogen bonds to the amino group of Lys-72 and protonation amido on the chiral carbon forms hydrogen bond with Ash-171 and forms salt bridge with Asp-184. This is the characteristic of binding mode between 3, 5-disubstituted pyridine inhibitor and protein kinase A/B. CoMFA and CoMSIA model present well prediction and the contour maps are taken as representative to explain factors affecting activities of inhibitors and provide the basis for further designing noval 3, 5-disubstituted pyridine inhibitor. According to the suggestion of CoMFA and CoMSIA model, it is helpful that a positive and small substituent added to position 1 of quinoline, a negative substituent added to position 2 of indole, and the carbon atom at position 4 of indole replaced by negative atom. According to result of the molecular dynamics simulation study that there is electrostatic repulsion between oxygen atom of inhibitor and carboxyl oxygen atom, replacement of the oxygen atom by amide or methylene might be helpful for activities.The information obtained from this study can help to know more about the structural characteristic of protein kinase A and further supports the point that the structural water molecule plays a crucial role in the biomolecule. The difference between the active site of protein kinase A and protein kinase C is detected and the rule in designing and optimizing 3, 5-disubstituted pyridine inhibitor is seek, which could provide instructive information for the structure-based drug design. It may also serve as a guide to studies of the other similar proteins.