Study On The Recognition And Interactions Of HIV-1TAR RNA With Cyclic Peptide Inhibitors And Related Proteins Via Molecular Modeling

Protein-RNA interactions are of great significance for many cellular regulationprocesses as well as viral replication. Therefore, the study of recognition mechanismsbetween them is a hot spot in the filed of life science. Many biochemical andbipphysical techniques have been employed to study the interactions between proteinand RNA. At the same time, the theoretical approaches also provide significantinformation. Molecular docking and molecular dynamics simulation, for instance,have been extensively and successfully applied to the interpretation and prediction ofstructure-function relationship of biological macromolecules.The HIV (Human Immunodeficiency Virus)-encoded trans-activator (Tat) proteinbinds directly to the Cyclin T1subunit of cellular positive transcription elongationfactor (P-TEFb), which provokes the recruitment of P-TEFb to the trans-activationresponse (TAR) RNA that is located at the5’-end of all nascent HIV-1transcripts.The other subunit CDK9of activated P-TEFb hyperphosphates the C-terminal domain(CTD) of RNA polymerase II (RNA Pol II), which greatly enhances the progressivityof RNA Pol II elongation complex, marking the transition from the transcriptioninitiation to transcription elongation. Hence, interfering with the complex formationof P-TEFb-Tat-TAR has long been viewed as an effective strategy against the HIVinfection.The main contents of this dissertation include the following two parts:1. Study on the interactions and recognition of cyclic peptide mimetics of Tatprotein with HIV-1TAR RNA via molecular dynamics simulationsIn this work, we chose two representative cyclic peptides named L-22andKP-Z-41with potent inhibitory activity against Tat-TAR interaction, and detected theinteractions and recognition between them and HIV-1TAR RNA using MDsimulations. The binding modes of these two peptides to TAR RNA are nearlyidentical at the inter-junction, whereas the binding interfaces at the RNA apical loopexhibit large conformational heterogeneity. From the results of principal componentanalysis, it was found that the intrinsic twisting motion within the free TAR RNA issignificantly constrained upon binding to the cyclic peptides, which may account forthe source of high activity for this class of peptidomimetics. In addition, we revealedthe electrostatic interaction energy contributes much more to KP-Z-41complexformation that to L-22complex, which is partially ascribed to the two P-end residuesArg1and Arg15in KP-Z-41. This is the main origin of energy that results in a higheraffinity of KP-Z-41than L-22for TAR RNA. Furthermore, we identified a conservedmotif RRK that is shown to be essential for the high affinity and selective recognitionof TAR RNA by this class of lead peptides. The results of this study could providesome implications for the improvement of Tat peptidomimetic inhibitors. 2. Modelling and optimizing the complex structure of hCycT1-hTat-hTARCurrently, the vast majority of compounds that prevent the complex formation ofthe human CycT1(hCycT1) with HIV-1Tat and TAR RNA are Tat-TAR interactioninhibitors. So far, the three dementional structure of hCycT1-hTat-hTAR complex hasnot yet been determined, which hinders the drug development aimed at the interactionregions within the ternary complex of hCycT1-hTat-hTAR. In this work, firstly weconstructed the heterodimer of hCycT1and hTat via homology modelling andmolecular superimposition. Secondly, we obtained tens of thousands of binding posesof hCycT1-hTat with hTAR by molecular docking approach, and one structure amongthem was screened out based on the clustering analysis in combination withexperimental information. Finally, the selected binding mode was optimized via MDsimulation. Encouragingly, the optimized complex structure is in good accordancewith available experimental data. The structure of hCycT1-hTat-hTAR presented hereis of help for the future research on the design of the inhibitors targeting theinteractions within the transcriptional elongation activation complex.