Saturday, February 18, 2017
Exhibit Hall (Hynes Convention Center)
Prateek Sharma, University of Michigan, Ann Arbor, MI
BACKGROUND: Phosphatidylinositol 3,4,5- trisphosphate (PIP3)-dependent Rac exchanger 1 (P- Rex1) is a Rho guanine-nucleotide exchange factor (RhoGEF) that regulates cytoskeletal rearrangement and cell motility and is linked to tumor metastasis. The P-Rex family includes P-Rex1 and P-Rex2, which are activated by both the lipid PIP3, generated by activated receptor tyrosine kinases, and free Gβγ subunits, generated by activated GPCRs. However, the structural basis behind functional regulation with P-Rex is poorly understood. My objective is to determine the structure of P-Rex2 PH and characterize the interaction of small molecules with different PH domains through qualitative analysis with differential scanning fluorimetry (DSF) and detailed assessment through X-ray crystallography. METHODS: Rosetta (DE3) pLysS E. coli (Novagen) cells were used to overexpress P-Rex2 PH constructs as N-terminally 10xHis-tagged maltose binding protein (MBP)-fusion proteins, which increases yield and facilitates purification. Recombinant protein was purified using Ni-NTA resin chromatography followed by a cleavage of the MBP tag with TEV protease. Final purity results from size exclusion chromatography. A DSF assay was used to analyze the binding interaction between compounds and PH domains. RESULTS: Crystallographic studies led to a high-resolution structure (1.90 Å) of the independent P-Rex2 PH domain. A comparison between the P-Rex1 PH and P-Rex2 PH structures shows divergence in the β3/β4 loop, shown by our lab to play a role in non-specific membrane localization, and the β5/β6 loop, a likely protein-protein interaction site. Using DSF, we identified six small molecules from a high-throughput screen that bind the P-Rex1 PH domain. P-Rex2 and Akt PH (a kinase PH domain which has been successfully targeted with small molecule inhibitors) were used to further assess compound binding to a variety of PH domains. IP4, a soluble analog of PIP3, resulted in a 12.3 °C shift in Tmfor P-Rex1 PH and a 15.4 °C shift for P-Rex2. Compound #13 (20 μM) was most notable in causing a 15.5 °C shift for P-Rex1, 22.5 °C for P-Rex2, and 13.5 °C for Akt PH. CONCLUSIONS: We have determined the structure of the P-Rex2 PH domain and have analyzed binding of small molecules to P-Rex1 PH, P-Rex2 PH, and Akt PH. We are currently co-crystallizing P-Rex1 PH with these small molecules to verify that they target the PIP3-binding site. As PIP3 binding is necessary for P-Rex activation, small molecules can be developed to perturb this interaction, resulting in therapeutics with anti-metastatic properties.