Expression, purification and characterization of the RhoA-binding domain of human SHIP2 in E.coli.

Abstract

Human SH2-containing inositol 5-phosphatase 2 (SHIP2) is a multi-domain protein playing essential roles in various physiological and pathological processes. In cell polarization and migration, SHIP2 serves as a RhoA effector for manipulating the level of phosphatidylinositol 3,4,5-trisphosphate. The domain between SH2 and a potential PH-R domain of SHIP2 was suggested to bind with GTP-bound form of RhoA. However, the structure of this RhoA-binding domain (RBD) of SHIP2 and the mechanism for its binding with RhoA remain unknown. In this study, SHIP2118-298 and SHIP2176-298, two truncated proteins harboring the RBD were designed, expressed, and purified successfully in E.coli. Unexpectedly, both SHIP2118-298 and SHIP2176-298 were determined to exist as homo-dimers in solution by multi-angle light scattering. Circular dichroism spectra indicated that both proteins predominantly consisted of α-helix structure. Moreover, in pull-down experiments, both proteins could bind with GTP-bound RhoA and RhoAQ63L, a mutant mimicing the state of GTP-bound RhoA. Importantly, in silico analysis showed that the shorter truncation, SHIP2176-298, contained all ordered residues between the SH2 and the PH-R domain, and matched the RhoA effector motif 1 of PKN1 well in sequence alignment, suggesting that SHIP2176-298 is sufficient for further studies on the structure and RhoA binding of SHIP2. This work shortens and confirms the main region of SHIP2 interacting with RhoA, provides the method for sample preparation, and presents preliminary information for SHIP2-RBD structure, which will facilitate the comprehensive understanding of the structure and function of SHIP2.