RESUMO
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.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Proteínas Serina-Treonina Quinases/química , Fatores de Troca de Nucleotídeo Guanina Rho/química , Animais , Microscopia Crioeletrônica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Neoplasias Uveais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Carcinoma Hepatocelular/metabolismo , Microscopia Crioeletrônica/métodos , AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/ultraestrutura , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/ultraestrutura , Holoenzimas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Simulação de Dinâmica Molecular , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Espalhamento a Baixo Ângulo , Difração de Raios X/métodosRESUMO
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.
Assuntos
Membrana Celular , Fatores de Troca do Nucleotídeo Guanina , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosforilação , Domínios ProteicosRESUMO
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.
Assuntos
Descoberta de Drogas/métodos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células Cultivadas , Cristalografia por Raios X/métodos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Neutrófilos/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Peixe-ZebraRESUMO
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.
RESUMO
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.
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Microscopia Crioeletrônica , Fosfatos de Fosfatidilinositol/metabolismo , Conformação Proteica , Ligação ProteicaRESUMO
TGF-ß family ligands are involved in a variety of critical physiological processes. For instance, the TGF-ß ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-ß family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.
Assuntos
Proteínas Relacionadas à Folistatina/química , Modelos Moleculares , Miostatina/química , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Proteínas Relacionadas à Folistatina/genética , Proteínas Relacionadas à Folistatina/metabolismo , Humanos , Miostatina/genética , Miostatina/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Myostatin is a member of the transforming growth factor-beta (TGF-beta) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-beta class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.
Assuntos
Folistatina/química , Heparina/metabolismo , Miostatina/química , Ativinas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Heparina/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300â keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200â keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200â keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9â Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8â Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200â keV.
RESUMO
Single-particle cryoelectron microscopy (cryo-EM) continues to grow into a mainstream structural biology technique. Recent developments in data collection strategies alongside new sample preparation devices herald a future where users will collect multiple datasets per microscope session. To make cryo-EM data processing more automatic and user-friendly, we have developed an automatic pipeline for cryo-EM data preprocessing and assessment using a combination of deep-learning and image-analysis tools. We have verified the performance of this pipeline on a number of datasets and extended its scope to include sample screening by the user-free assessment of the qualities of a series of datasets under different conditions. We propose that our workflow provides a decision-free solution for cryo-EM, making data preprocessing more generalized and robust in the high-throughput era as well as more convenient for users from a range of backgrounds.
Assuntos
Microscopia Crioeletrônica/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Crioeletrônica/normas , Aprendizado Profundo , Ensaios de Triagem em Larga Escala/normas , Processamento de Imagem Assistida por Computador/normas , Conformação ProteicaRESUMO
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 1.9â¯Å crystal 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.
RESUMO
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.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Microscopia Crioeletrônica/métodos , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Alinhamento de SequênciaRESUMO
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.
Assuntos
Domínio Catalítico , Fatores de Troca do Nucleotídeo Guanina/química , Fosfatos de Fosfatidilinositol/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Myostatin, a member of the transforming growth factor (TGF)-ß family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-ß family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-ß family.
Assuntos
Ativinas/antagonistas & inibidores , Substâncias de Crescimento/farmacologia , Miostatina/antagonistas & inibidores , Simulação por Computador , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Bibliotecas de Moléculas PequenasRESUMO
Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-ß family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-ß family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.