Structural And Functional Study Of The RhoA Binding Domain Of Human SHIP2 Protein

In living organisms,cells are not static,The development of organisms are accompanied by a large number of cell migration events.Most of the migrating cells have a highly polarized morphology,and the polarization of the cells is precisely regulated by a variety of signaling molecules,such as phosphatidylinositol（Ptd Ins）and the small GTPases RhoA.Phosphatidylinositol phosphatase SHIP2 catalyzes the hydrolysis of Ptd Ins（3,4,5）P3 into Ptd Ins（3,4）P2,and can interact with RhoA to reduce Ptd Ins（3,4,5）P in the cell membrane at the back of the cell The content affects the transmission of Ptd Ins-related cell signals,and then regulates cell local polarization and cell migration,which is closely related to human development and tumor development.Structure determines function,and studying protein structure is crucial for understanding the mechanism by which it functions.Studying the structure of the RhoA-binding domain（RBD）of SHIP2 and the interaction molecular mechanism between SHIP2 and RhoA will help to fully understand the function of SHIP2 and its related processes such as human development and tumorigenesis.we selected the region of human SHIP2 protein that interacts with RhoA as the research object.The main research contents and results are as follows:1.Design,preparation and characterization of the truncated SHIP2 proteins.Disordered/ordered structure analysis of SHIP2 using online software Pr DOS,the sequence comparison with the known structure of the RhoA interaction protein PKN^1-100,and the domain distribution of SHIP2,two SHIP2 truncations were designed,SHIP2^118-298and SHIP2^176-298,which were expressed and purified through the E.coli system,and obtained high-yield and high-purity protein samples,which were confirmed by mass spectrometry.Static light scattering experiments show that SHIP2^118-298and SHIP2^176-298proteins mainly exist as homodimers in solution.The circular dichroism experiment showed that both SHIP2^118-298and SHIP2^176-298proteins containedα-helical secondary structure,which mainly existed in the region of176-298 amino acid residues.Only the chemical shift peaks of the residues in the disordered structure region were observed in the liquid NMR spectrum,indicating that the conformation of the ordered structure region in SHIP2^118-298and SHIP2^176-298proteins is unstable and there may be dynamic conformational exchange.2.Interaction between truncated SHIP2 proteins and RhoA.2.The interaction between SHIP2 truncated body protein and RhoA protein.In this study,various forms of RhoA protein samples were prepared,including wild-type RhoA,GDP-bound and GTPγS（GTP steady-state analogs）bound samples,and RhoA^Q63Lmutant samples that mimic the activated state conformation.Pull down experiments showed that both SHIP2^118-298and SHIP2^176-298can interact with GTPγS-bound RhoA and RhoA^Q63Lmutants,confirming the interaction between SHIP2 and activated RhoA,and indicating that the SHIP2 protein interval containing176-298 residues is sufficient to interact with The interaction of RhoA is the main area of interaction.In conclusion,in this study,two truncated SHIP2 proteins were successfully prepared for structural and functional studies,and it was found that SHIP2 mainly existed in the form of homodimer.On the basis of previous studies,we verified that SHIP2 could bind to RhoA activated state,revealed the major interaction region between SHIP2 and RhoA was located in the residues 176-298,and found that this region mainly containedα-helix structure.This study shortened and confirmed the scope of SHIP2 RBD,established sample preparation methods for the RBD,and provided its preliminary structure information,which lays a solid foundation for the subsequent structure and function of SHIP2-RBD,and will promote the understanding of SHIP2 function and its related human development processes and diseases.