P-Rex2 exhibits unique structural features and regulatory mechanisms distinct from the closely related RhoGEF P-Rex1

Abstract Rho guanine-nucleotide exchange factors (RhoGEFs) activate small GTPases to drive cytoskeletal rearrangement, cell motility, and proliferation. The phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) subfamily of RhoGEFs includes P-Rex1 and P-Rex2 which, when misregulated, contribute to cancer progression and metastasis. P-Rex activity is controlled by accessory domains that maintain the protein in a cytosolic, autoinhibited state until activated by the lipid PIP3 and G protein βγ subunits. While P-Rex1 autoinhibition has been structurally and biochemically characterized, P-Rex2 has remained largely unexplored. Furthermore, despite high sequence similarity and domain conservation, P-Rex homologs differ in substrate specificity and regulatory interactions, and the molecular basis for these divergences is unknown. Here, we have taken an integrative structural biology approach to investigate these gaps. Using cryo-EM, we determined the first structure of full-length P-Rex2 to moderate resolution, revealing that, while the overall structure closely resembles that of P-Rex1, there is a substantial repositioning of the N-terminal module relative to the C-terminal core. This may play a key role in precluding the intramolecular interactions between the N- and C-terminal domains that are observed in autoinhibited P-Rex1. Hydrogen-deuterium exchange mass spectrometry revealed that, unlike P-Rex1, P-Rex2 dynamics are unaffected by IP4, the headgroup of PIP3. SEC-SAXS data support that the N-terminal module itself is less dynamic, and biochemical assays show that P-Rex2 may be more tightly regulated by autoinhibition, likely through a mechanism different from P-Rex1. These findings uncover unique features in the molecular mechanisms of P-Rex2 regulation. Competing Interest Statement The authors have declared no competing interest. Abbreviations and nomenclature - RhoGEF - Rho guanine-nucleotide exchange factor - PIP3 - phosphatidylinositol-3,4,5-trisphosphate - P-Rex - phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchanger - GDP - guanosine diphosphate - GTP - guanosine triphosphate - Rac - Ras-related C3 botulinum toxin - Cdc42 - cell division control protein 42 homolog - Dbl - diffuse B-cell lymphoma - DH - Dbl homology - PH - pleckstrin homology - DEP - Dishevelled, Egl-10 and Pleckstrin - PDZ - Postsynaptic density protein 95, Discs large protein, and Zonula occludens-1 - IP4P - inositol polyphosphate-4-phosphatase-like - PI3K - phosphoinositide 3-kinases - 4HB - 4-helix bundle - IP4 - inositol-(1,3,4,5)-tetrakisphosphate - PTEN - phosphatase and tensin homolog - cryo-EM - cryo-electron microscopy - HDX-MS - hydrogen-deuterium exchange mass spectrometry - XL-MS - cross-linking mass spectrometry - SEC-SAXS - small-angle X-ray scattering - FL - full-length - RMSD - root mean squared deviation - COMs - centers of mass - SDS-PAGE - sodium dodecyl sulfate–polyacrylamide gel electrophoresis - mant-GTP - N-methyl-anthraniloyl-GTP - P(r) - pair distance distribution - EOM - employed ensemble optimization method - WT - wild-type - MBP - maltose binding protein - GST - glutathione S-transferase - DTT - dithiothreitol - HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - NaCl - sodium chloride - EDTA - ethylenediaminetetraacetic acid - CV - column volumes - IPTG - isopropyl β-D-1-thiogalactopyranoside - MWCO - molecular weight cutoff - DDM - n-Dodecyl-Beta-Maltoside - PNCC - Pacific Northwest Center for Cryo-EM - BIS - beam image shift - CTF - contrast transfer function - FSC - fourier shell correlation - GuHCl - guanidine hydrochloride - LC/MS - liquid chromatography/mass spectrometry - D2O - deuterium oxide - Tris - Tris(hydroxymethyl)aminomethane - MgCl2 - magnesium chloride - Rg - radius of gyration - Vc - volume of correlation