Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4.

PIP3-dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and heterotrimeric Gßγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP3, we discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4.

PIP3-dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and the heterotrimeric Gßγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP3, we discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

Structural/functional studies of Trio provide insights into its configuration and show that conserved linker elements enhance its activity for Rac1.

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.

Structural analyses of the PKA RIIß holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIß, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIß:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIß2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIß holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIß holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity and contributes to membrane binding.

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gßγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

Discovery of Small Molecules That Target the Phosphatidylinositol (3,4,5) Trisphosphate (PIP3)-Dependent Rac Exchanger 1 (P-Rex1) PIP3-Binding Site and Inhibit P-Rex1-Dependent Functions in Neutrophils.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein ßγ subunits and the lipid PIP3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.

Cryo-electron microscopy structure and analysis of the P-Rex1-Gßγ signaling scaffold.

PIP3-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1-Gßγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gßγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gßγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gßγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.

Structural and biochemical characterization of the pleckstrin homology domain of the RhoGEF P-Rex2 and its regulation by PIP3.

P-Rex family Rho guanine-nucleotide exchange factors are important regulators of cell motility through their activation of a subset of small GTPases. Both P-Rex1 and P-Rex2 have also been implicated in the progression of certain cancers, including breast cancer and melanoma. Although these molecules display a high level of homology, differences exist in tissue distribution, physiological function, and regulation at the molecular level. Here, we sought to compare the P-Rex2 pleckstrin homology (PH) domain structure and ability to interact with PIP3 with those of P-Rex1. The 1.9 Šcrystal structure of the P-Rex2 PH domain reveals conformational differences in the loop regions, yet biochemical studies indicate that the interaction of the P-Rex2 PH domain with PIP3 is very similar to that of P-Rex1. Binding of the PH domain to PIP3 is critical for P-Rex2 activity but not membrane localization, as previously demonstrated for P-Rex1. These studies serve as a starting point in the identification of P-Rex structural features that are divergent between isoforms and could be exploited for the design of P-Rex selective compounds.

Structural and Biochemical Characterization of the Catalytic Core of the Metastatic Factor P-Rex1 and Its Regulation by PtdIns(3,4,5)P3.

Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine nucleotide exchange factor synergistically activated by PIP3 and Gßγ that plays an important role in the metastasis of breast, prostate, and skin cancer, making it an attractive therapeutic target. However, the molecular mechanisms behind P-Rex1 regulation are poorly understood. We determined structures of the P-Rex1 pleckstrin homology (PH) domain bound to the headgroup of PIP3 and resolved that PIP3 binding to the PH domain is required for P-Rex1 activity in cells but not for membrane localization, which points to an allosteric activation mechanism by PIP3. We also determined structures of the P-Rex1 tandem Dbl homology/PH domains in complexes with two of its substrate GTPases, Rac1 and Cdc42. Collectively, this study provides important molecular insights into P-Rex1 regulation and tools for targeting the PIP3-binding pocket of P-Rex1 with a new generation of cancer chemotherapeutic agents.