RESUMO
The controlled derivatization of natural products is of great importance for their use in drug discovery. The ideally rapid generation of compound libraries for structure-activity relationship studies is of particular concern. We here use modified biosynthesis for the generation of such a library of reduced polyketides to interfere with the oncogenic KRas pathway. The polyketide is derivatized via side chain alteration, and variations in its redox pattern and in its backbone chain length through manipulation in the corresponding polyketide synthase. Structural and biophysical analyses revealed the nature of the interaction between the polyketides and KRas-interacting protein PDE6δ. Non-natural polyketides with low nanomolar affinity to PDE6δ were identified.
Assuntos
Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Humanos , Policetídeo Sintases/química , Policetídeos/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-AtividadeRESUMO
Translationally controlled tumour protein (TCTP) is involved in malignant transformation and regulation of apoptosis. It has been postulated to serve as a guanine nucleotide exchange factor for the small G-protein Rheb. Rheb functions in the PI3 kinase/mTOR pathway. The study presented here was initiated to characterise the interaction between TCTP and Rheb biochemically. Since (i) no exchange activity of TCTP towards Rheb could be detected in vitro, (ii) no interaction between TCTP and Rheb could be detected by NMR spectroscopy, and (iii) no effect of TCTP depletion in cells on the direct downstream targets of Rheb could be observed in vivo, this study shows that TCTP is unlikely to be a guanine nucleotide exchange factor for Rheb.
Assuntos
Biomarcadores Tumorais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/genética , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Interferência de RNA , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
GidA is a flavin-adenine-dinucleotide (FAD)-binding protein that is conserved among bacteria and eucarya. Together with MnmE, it is involved in the addition of a carboxymethylaminomethyl group to the uridine base in the wobble position (nucleotide 34) of tRNAs that read split codon boxes. Here, we report the crystal structures of the GidA proteins from both Escherichia coli and Chlorobium tepidum. The structures show that the protein can be divided into three domains: a first FAD-binding domain showing the classical Rossmann fold, a second alpha/beta domain inserted between two strands of the Rossmann fold, and an alpha-helical C-terminal domain. The domain inserted into the Rossmann fold displays structural similarity to the nicotinamide-adenine-dinucleotide-(phosphate)-binding domains of phenol hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, and, correspondingly, we show that GidA binds NADH with high specificity as an initial donor of electrons. GidA behaves as a homodimer in solution. As revealed by the crystal structures, homodimerization is mediated via both the FAD-binding domain and the NADH-binding domain. Finally, a large patch of highly conserved, positively charged residues on the surface of GidA leading to the FAD-binding site suggests a tRNA-binding surface. We propose a model for the interaction between GidA and MnmE, which is supported by site-directed mutagenesis. Our data suggest that this interaction is modulated and potentially regulated by the switch function of the G domain of MnmE.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , RNA de Transferência/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Chlorobium/química , Cristalografia por Raios X , Dimerização , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Histidina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , NADP/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA de Transferência/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. Here we present the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its 'switch' regions for interacting with two subdomains of GBD/FH3. We show that the FH3 domain of mDia1 forms a stable dimer and we also identify the DAD-binding site. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of our results, we propose a structural model for the regulation of mDia1 by Rho and DAD.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Dimerização , Forminas , Células HeLa , Humanos , Mamíferos , Modelos Moleculares , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Guanine nucleotide-binding proteins regulate a variety of processes, including sensual perception, protein synthesis, various transport processes, and cell growth and differentiation. They act as molecular switches and timers that cycle between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Recent structural studies show that the switch apparatus itself is a conserved fundamental module but that its regulators and effectors are quite diverse in their structures and modes of interaction. Here we will try to define some underlying principles.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Regulação Alostérica , Sítios de Ligação , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Hidrólise , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: The small GTP binding protein Ras has important roles in cellular growth and differentiation. Mutant Ras is permanently active and contributes to cancer development. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase. The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms. RESULTS: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components. The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization. A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region. CONCLUSIONS: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.
Assuntos
Proteínas Fúngicas/química , MAP Quinase Quinase Quinases , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimologia , Proteínas ras/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Proteínas ras/metabolismoRESUMO
p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Animais , Catálise , Domínio Catalítico , Cromatografia em Gel , Dicroísmo Circular , Dimerização , Relação Dose-Resposta a Droga , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Glutationa Transferase/metabolismo , Cinética , Modelos Biológicos , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Espectrometria de Fluorescência , Fatores de Tempo , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21 , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Catálise , Escherichia coli , Genes ras , Ligação Proteica , Especificidade por SubstratoRESUMO
For the study of biological signal transduction, access to correctly lipidated proteins is of utmost importance. Furthermore, access to bioconjugates that embody the correct structure of the protein but that may additionally carry different lipid groups or labels (i.e., fluorescent tags) by which the protein can be traced in biological systems, could provide invaluable reagents. We report here of the development of techniques for the synthesis of a series of modified Ras proteins. These modified Ras proteins carry a number of different, natural and non-natural lipid residues, and the process was extended to also provide access to a number of fluorescently labeled derivatives. The maleimide group provided the key to link chemically synthesized lipopeptide molecules in a specific and efficient manner to a truncated form of the H-Ras protein. Furthermore, a preliminary study on the biological activity of the natural Ras protein derivative (containing the normal farnesyl and palmitoyl lipid residues) has shown full biological activity. This result highlights the usefulness of these compounds as invaluable tools for the study of Ras signal transduction processes and the plasma membrane localization of the Ras proteins.
Assuntos
Corantes Fluorescentes/química , Lipoproteínas/síntese química , Proteínas ras/síntese química , Animais , Eletroforese em Gel de Poliacrilamida , Lipoproteínas/química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Células PC12 , Ratos , Análise Espectral , Proteínas ras/químicaRESUMO
The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of the metal ion in the inactive, GDP-bound Ras has been studied by continuous wave EPR at 94 GHz (W-band). Via replacement of normal water with (17)O-enriched water, the (17)O--(55)Mn superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In contrast to EPR data on frozen solutions and X-ray data from single crystals where four direct ligands to the metal ion are found, the wild-type protein has only three water ligands bound in solution at room temperature. The same number of water ligands is found for the mutant Ras(T35S). However, for the alanine mutant in position 35 Ras(T35A) as well as for the oncogenic mutant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by (31)P NMR studies on the Mg(2+) x GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two conformational states (1 and 2) with an equilibrium constant K(1)(1,2) of approximately 0.49 and rate constants k(1--1) which are much smaller than 40 s(-1) at 298 K. For wild-type Ras and Ras(T35S), the two states can also be observed with equilibrium constants K(1)(1,2) of approximately 0.31 and 0.21, respectively. In Ras(G12V), only one conformational state could be detected.
Assuntos
Guanosina Difosfato/química , Manganês/química , Água/química , Proteínas ras/química , Substituição de Aminoácidos/genética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular/métodos , Mutação Puntual , Soluções , Temperatura , Proteínas ras/genéticaRESUMO
RCC1 (regulator of chromosome condensation), a beta propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran*RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran*RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.
Assuntos
Sítios de Ligação , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/química , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas Nucleares/química , Estrutura Terciária de Proteína , Proteína ran de Ligação ao GTP/química , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polieletrólitos , Polímeros/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Termodinâmica , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Calorimetria , Entropia , Modelos Moleculares , Mutação , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína da Síndrome de Wiskott-Aldrich , Proteínas ral de Ligação ao GTP/química , Proteínas ral de Ligação ao GTP/metabolismoRESUMO
We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)(.)GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. (31)P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. (31)P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.
Assuntos
Guanilil Imidodifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Treonina/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação , Sequência Conservada/genética , Cristalografia por Raios X , Guanilil Imidodifosfato/química , Humanos , Isomerismo , Cinética , Ligantes , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/genética , Treonina/genéticaRESUMO
Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.
Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sítios de Ligação , AMP Cíclico/análogos & derivados , Fluorescência , Produtos do Gene vpr/metabolismo , Vetores Genéticos , Glutationa Transferase/metabolismo , Marcação por Isótopo , Cinética , Estrutura MolecularRESUMO
ExoS is a bifunctional type III cytotoxin that is secreted by Pseudomonas aeruginosa. The N-terminal domain comprises a RhoGAP activity, while the C-terminal domain comprises a ADP-ribosyltransferase activity. Previous studies showed that ExoS ADP ribosylated Ras at Arg41 which interfered with the ability of Ras to interact with its guanine nucleotide exchange factor. Rap and Ras share considerable primary amino acid homology, including Arg41. In this study, we report that ExoS ADP ribosylates Rap1b at Arg41 and that ADP ribosylation of Arg41 inhibits the ability of C3G to stimulate guanine nucleotide exchange. The mechanism responsible for this inhibition is one in which ADP-ribosylated Rap binds inefficiently to C3G, relative to wild type Rap. This identifies a second member of the Ras GTPase subfamily that can be ADP ribosylated by ExoS and indicates that ExoS can inhibit both Ras and Rap signaling pathways in eukaryotic cells.
Assuntos
ADP Ribose Transferases , Adenosina Difosfato Ribose/metabolismo , Arginina/metabolismo , Toxinas Bacterianas , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Proteínas Quinases/metabolismo , Proteínas rap de Ligação ao GTP/antagonistas & inibidores , Proteínas rap de Ligação ao GTP/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/antagonistas & inibidores , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Histidina/genética , Histidina Quinase , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas rap de Ligação ao GTP/genéticaRESUMO
Pseudomonas aeruginosa is an opportunistic bacterial pathogen of great medical relevance. One of its major toxins, exoenzyme S (ExoS), is a dual function protein with a C-terminal Ras-ADP-ribosylation domain and an N-terminal GTPase activating protein (GAP) domain specific for Rho-family proteins. We report here the three-dimensional structure of the N-terminal domain of ExoS determined by X-ray crystallography to 2.4 A resolution. Its fold is all helical with a four helix bundle core capped by additional irregular helices. Loops that are known to interact with Rho-family proteins show very large mobility. Considering the importance of ExoS in Pseudomonas pathogenicity, this structure could be of interest for drug targeting.
Assuntos
ADP Ribose Transferases/química , Proteínas de Bactérias/química , Toxinas Bacterianas , Proteínas Ativadoras de GTPase/química , Pseudomonas aeruginosa/enzimologia , Cristalografia por Raios X , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/químicaRESUMO
Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Regulação para Baixo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas rac de Ligação ao GTP/metabolismo , Compostos de Alumínio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Fluoretos/metabolismo , Proteínas Ativadoras de GTPase/química , Guanosina Difosfato/metabolismo , Histidina Quinase , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/patogenicidade , Alinhamento de Sequência , Relação Estrutura-Atividade , Proteínas rac de Ligação ao GTP/químicaRESUMO
The small guanosine triphosphate (GTP) binding protein Ras is involved in many cellular signal transduction processes leading to cell growth, differentiation and apoptosis. Mutations in ras genes are found in a large number of human tumours. GTP hydrolysis, the process that normally leads to the transition of the Ras protein from the active (GTP-bound) form to the inactive (GDP-bound) form is impaired due to these oncogenic mutations. In contrast, the GTP analogue 3,4-diaminobenzophenone(DABP)-phosphoramidate-GTP, a substrate for GTP-binding proteins, enables switching to the inactive GDP form in both wild-type and oncogenic Ras. Here we show by HPLC, mass spectrometry and NMR spectroscopy that the mechanism of this DABP-GTPase reaction is different from the physiological GTPase reaction. The gamma-phosphate group is not attacked by a nucleophilic water molecule, but rather by the aromatic amino group of the analogue, which leads to the generation of a stable cyclic diamidate product. These findings have potential implications for the development of anti-Ras drugs.
Assuntos
Benzofenonas/química , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Proteínas ras/química , Benzofenonas/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Ressonância Magnética Nuclear Biomolecular , Proteínas ras/metabolismoRESUMO
Mutations of Ras with three extra amino acids inserted into the phosphate-binding (P) loop have been investigated both in vitro and in vivo. Such mutants have originally been detected as oncogenes both in the ras and the TC21 genes. Biochemical experiments reveal the molecular basis of their oncogenic potential: the mutants show a strongly attenuated binding affinity for nucleotides, most notably for GDP, leading to a preference for GTP binding. Furthermore, both the intrinsic as well as the GAP-stimulated GTP hydrolysis are drastically diminished. The binding interaction with GAP is reduced, whereas binding to the Ras-binding domain of the downstream effector c-Raf1 is not altered appreciably. Microinjection into PC12 cells shows the mutants to be as potent to induce neurite outgrowth as conventional oncogenic Ras mutants. Unexpectedly, their ability to stimulate the MAP kinase pathway as measured by a reporter gene assay in RK13 cells is much higher than that of the normal oncogenic mutant G12V. This characteristic was attributed to an increased stimulation of c-Raf1 kinase activity by the insertional Ras mutants.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Clonagem Molecular , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Genes ras , Mutagênese Insercional , Nucleotídeos , Proteína Oncogênica p21(ras)/genética , Células PC12 , Fosfatos/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Coelhos , RatosRESUMO
The interferon-gamma-induced guanylate-binding protein 1 (GBP1) belongs to a special class of large GTP- binding proteins of 60-100 kDa with unique characteristics. Here we present the structure of human GBP1 in complex with the non-hydrolysable GTP analogue GppNHp. Basic features of guanine nucleotide binding, such as the P-loop orientation and the Mg(2+) co-ordination, are analogous to those of Ras-related and heterotrimeric GTP-binding proteins. However, the glycosidic bond and thus the orientation of the guanine base and its interaction with the protein are very different. Furthermore, two unique regions around the base and the phosphate-binding areas, the guanine and the phosphate caps, respectively, give the nucleotide-binding site a unique appearance not found in the canonical GTP-binding proteins. The phosphate cap, which constitutes the region analogous to switch I, completely shields the phosphate-binding site from solvent such that a potential GTPase-activating protein cannot approach. This has consequences for the GTPase mechanism of hGBP1 and possibly of other large GTP-binding proteins.