Structure And Function Of Tenisn2SH2Domain From Homo Sapiens And The ADF/cofilin From Trypanosoma Brucei

This dissertation focuses on the structure and function of tensin2SH2domain from Homo sapiens and the ADF/cofilin from Trypanosoma brucei.(I) Src homology2(SH2) domain is a conserved module involved in various biological processes. Tensin family proteins localize to focal adhesions of cells. All the members of tensin family are proteins with multidomain, which allow tensins to interact with several ligands simultaneously. This property of tensin family reflects its essential role in signal transduction pathway. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1(deleted-in-liver-cancer-1) via its SH2domain. In this study, we determined the solution structure of tensin2SH2domain from Homo sapiens by Nuclear Magnetic Resonance (NMR) spectroscopy. Tensin2SH2domain adopted a typical SH2fold that mainly consists of five (3-strands at the centre and flanked by two a-helices. Most SH2domains recognize phosphorylated ligands specifically. However, tensin2SH2domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand. Furthermore, chemical shift perturbation was performed to identify the residues responsible for binding to tyrosine-phosphorylated or nonphosphorylated DLC-1peptide in tensin2SH2domain. The results indicated a similar manner of tensin2SH2domain in binding to nonphosphorylated and phosphorylated ligands. The tumor suppressive function of DLC-1relies on the interaction between DLC-1and the SH2domain of tensins. Therefore, our research on this interaction might provide a structural basis for the treatment of cancer.(II) As one of the most abundant and conserved cytoskeletal proteins in eukaryotic cells, actin is important for cell migration, intracellular transport, cell division and transcription regulation. The actin-depolymerizing factor (ADF)/cofilin family is one of the most important regulators of the spatial and temporal organization of actin filaments. In this study, we identify the ADF/cofilin from Trypanosoma brucei. The solution structure of the ADF/cofilin from Trypanosoma brucei (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central (3-sheet composed of six β-strands surrounded by five a-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof did not co-sediment with but depolymerized actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from Trypanosoma brucei. The identification of TbCof could benefit for further investigation on the kinetic regulation of actin-filaments in Trypanosoma brucei.