Speaker: Xiaowei Hoi, Senior Research Scientist - Memorial Sloan Kettering Cancer Center
Title: Structural studies reveal insights into CRAC channel activation
Abstract: Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) is a Rho guanine-nucleotide exchange factor that regulates cell motility through its activation of small GTPases such as Rac1 and Cdc42. P-Rex is synergistically recruited to the cell membrane and activated by PIP3 and Gβγ subunits, positioning the enzyme downstream of multiple classes of cell surface receptors including GPCRs and receptor tyrosine kinases (RTKs). P-Rex1 functions as a critical regulator of neutrophil migration, but aberrant upregulation of P-Rex1 is strongly associated with cancer metastasis, and as such it has become an attractive therapeutic target. Development of selective inhibitors against P-Rex1 is hindered by the fact that its overall architecture and regulatory mechanisms are poorly understood. Thus, we have taken a multi-pronged structural biology approach using X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and cryo-electron microscopy (EM) to define the molecular basis for the regulation of P-Rex1, with the aim of identifying its important regulatory surfaces and mechanisms of activation. We first determined crystal structures of the P-Rex1 tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core and showed that the PH domain is necessary and sufficient for PIP3-dependent activation. Our data are consistent with PIP3 activating P-Rex1 via an allosteric mechanism. Using these structures, along with data from HDX-MS and cell-based experiments, we developed a model for P-Rex regulation wherein another domain within P-Rex1 binds to the DH and PH domains, resulting in a condensed tertiary structure that occludes the substrate-binding site on the DH domain. To test this model, we are analyzing cryo-EM data of P-Rex1 alone and in complex with regulatory molecules with the goal of providing high-resolution information for the full-length, 183 kDa protein. To this end, we have generated a 3.2 Å resolution structure of the P-Rex1–Gβγ complex. This structure shows a condensed conformation for P-Rex1 consistent with our HDX-MS experiments. This work has led to great insight into how P-Rex1 is regulated at the cell membrane, dissipating controversies that have existed in the P-Rex field for over a decade. Furthermore, we have discovered that the C-terminal domain of P-Rex1 shares structural homology with unusual phosphatases from Legionella and exhibits PI-3-phosphatase activity, opening up intriguing possibilities for auto-regulation. By investigating P-Rex1 structure and regulation through a variety of complementary approaches, we hope to guide the future development of therapeutic molecules.