Research Into Structure Of Human Small GTPase-Rnd1and Its Interaction With PlexinB1

Rndl, Rnd2, Rnd3are signal transduction GTPase proteins, which belongs to the newly found sub-branch of Rho GTPase. They possess strikingly different functional properties from other members of the Rho family. They have low GDP binding affinity and lacks intrinsic GTPase activity. Foster et al. and Nobes et al. firstly revealed involvement of Rndl in neurite outgrowth, dendrite development, and axon guidance. But recently, abnormal Rndl expression was also found in gastric cancer and endothelial cells during inflammation. Rndl plays different roles in signaling through its interaction with multiple protein partners. Binding between Rndl and pelxinB1RBD gained the most attention. This was one of the firstly reported direct interactions between GTPase and transmembrane protein receptors. Through interaction with plexinB1, Rndl participated in Sema3D signaling transduction, and activated the activity of plexinBl towards R-Ras. The Rndl-plexinBl-Sema4D signaling pathway is highly related with cancer diseases. Abnormal expression and mutation of both Sema4D and plexinB1has been found in different cancer cell types. More importantly, several plexinB1mutations with high frequency occurred in the RBD (Rho GTPase binding domain) region which binds with Rndl.Structural studies are crucial for understanding the mechanism underlying protein functions and interactions. NMR and X ray are two principal complementary methods for probing structural and dynamic properties of macromolecules. Compared to X-Ray, NMR determines the structures of proteins in solution which more resemble physiological environment and has no necessity of crystallization. NMR also has unique advantage in dynamics research. It yields information on multiple timescales ranging from nanosecond, microsecond to second. Moreover, It can measure site-specific dynamic behavior.This thesis mainly utilized NMR techniques to study the Rndl structure in solution, and also the conformation and dynamics changes during its interaction with plexinB1RBD. These study included following major aspects:(1) Multiple approaches were utilized to enhance Rndl stability and improve its solubility in solution:cleavage of the His tag, screening of buffer conditions, addition of a non-hydrolysable GTP analogue, GMPPNP, and also site directed mutation. These efforts not only significantly improved stability and solubility of the protein, also the NMR spectra quality.(2) Rndl protein was prepared by isotopic labeling and amino acid specific15N labeling. Through combination of common multi-dimensional spectra for protein NMR assignment and the3D NOESY experiment, backbone assignment was accomplished for127resonances in the total196amino acids of Rndl protein. Based on the chemical shift values resulted from assignment, Rndl secondary structure was analyzed. The solution structure is similar to the crystal structure in general. But there are differences in specific regions.(3) Based on Rndl NMR assignment and structure analysis, experiments were performed to study the conformation and dynamics changes of Rndl during its interaction with plexinB1RBD. Results indicated that, both proteins retained their overall conformations after binding but they are obviously affected in some local regions. Results also revealed the compensatory feature of protein dynamic change in different regions, and the "hinge" regions of Rndl play a critical role in conformational and dynamics changes during association. Rndl and Racl shared many common features during their binding with plexinB1RBD. They associated with plexinB1RBD through similar regions, undergoing resembling dynamic changes.(4) Study results of Rndl structure and its interaction with plexinB1was utilized for structure based design. Using computer aided virtual drug design, a batch of compounds having potential inhibitory activity was found. Meanwhile, previously reported Racl inhibitors were applied to Rndl for trial. Experiments first measured their binding ability with Rndl. The inhibitors then were further tested for their effect on the interaction between Rndl and plexinB1RBD. Preliminary results indicated one of the inhibitors NSC23766can bind Rnd1potentially。The NMR assignment and structure analysis in this thesis paves the way for further Rndl NMR study, especially study of Rndl interaction with other proteins. It also provides a guide for optimization and stabilization of other Rho GTPases. Study about interaction between Rndl and plexin-B1, not only uncovered the mechanism of Rndl functions, but also set a robust basis for structure based drug design targeting Rndl and plexinB1RBD. The preliminary small molecules screening made Rndl inhibitors exploration one step forward.