RESUMO
Uncovering molecular basis with regard to the conformational change of two switches I and II in the GppNHp (GNP)-bound H-Ras is highly significant for the understanding of Ras signaling. For this purpose, accelerated molecular dynamics (aMD) simulations and principal component (PC) analysis are integrated to probe the effect of mutations G12V, T35S and Q61K on conformational transformation between two switches of the GNP-bound H-Ras. The RMSF and cross-correlation analyses suggest that three mutations exert a vital effect on the flexibility and internal dynamics of two switches in the GNP-bound H-Ras. The results stemming from PC analysis indicate that two switches in the GNP-bound WT H-Ras tend to form a closed state in most conformations, while those in the GNP-bound mutated H-Ras display transformation between different states. This conclusion is further supported by free energy landscapes constructed by using the distances of residues 12 away from 35 and 35 away from 61 as reaction coordinates and different experimental studies. Interaction scanning is performed on aMD trajectories and the information shows that conformational transformations of two switches I and II induced by mutations extremely affect the GNP-residue interactions. Meanwhile, the scanning results also signify that residues G15, A18, F28, K117, A146 and K147 form stable contacts with GNP, while residues D30, E31, Y32, D33, P34 and E62 in two switches I and II produce unstable contacts with GNP. This study not only reveals dynamic behavior changes of two switches in H-Ras induced by mutations, but also unveils general principles and mechanisms with regard to functional conformational changes of H-Ras.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/química , Guanilil Imidodifosfato/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Maleabilidade , Análise de Componente Principal , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , TermodinâmicaRESUMO
Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/química , Acetilação , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Magnésio/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu ("EF-Pyl"). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg(2+), MM1309·GDP·Mg(2+), and MM1309·GMPPNP·Mg(2+) provided dissociation constants of 0.43, 26.2, and 222.2 µM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain-domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNA(Cys) and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNA(Cys).
Assuntos
Proteínas Arqueais/química , Guanosina Trifosfato/química , Methanosarcina/química , RNA de Transferência de Cisteína/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Calorimetria , Cristalografia por Raios X , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/química , Guanilil Imidodifosfato/metabolismo , Cinética , Methanosarcina/genética , Methanosarcina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Transferência de Cisteína/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.
Assuntos
Guanilil Imidodifosfato/química , NADPH Oxidases/química , Oryza/química , Fosfatos de Fosfatidilinositol/química , Proteínas de Plantas/química , Proteínas rac1 de Ligação ao GTP/química , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Guanilil Imidodifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oryza/enzimologia , Oryza/genética , Oryza/imunologia , Oxirredução , Fosfatos de Fosfatidilinositol/metabolismo , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The small GTPase Ras is an important signaling molecule acting as a molecular switch in eukaryotic cells. Recent findings of global conformational exchange and a putative allosteric binding site in the G domain of Ras opened an avenue to understanding novel aspects of Ras function. To facilitate detailed NMR studies of Ras in physiological solution conditions, we performed backbone resonance assignments of Ras bound to slowly hydrolysable GTP mimic, guanosine 5'-[ß, γ-imido]triphosphate at pH 7.2. Out of 163 non-proline residues of the G domain, signals from backbone amide proton, nitrogen and carbon spins of 127 residues were confidently assigned with the remaining unassigned residues mostly located at the exchange-broadened effectors interface.
Assuntos
Guanilil Imidodifosfato/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Concentração de Íons de Hidrogênio , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The peptide corticotropin-releasing factor (CRF) binds to the CRF1 receptor via a two-domain mechanism such that the extracellular domain (ECD) of the receptor captures the CRF's C-terminus to facilitate the binding of CRF's N-terminus to the juxta-membrane or "J"-site. Known small molecule antagonists bind to the J-site while known CRF1 receptor peptide radioligands bind to both sites. We report here the in vitro binding properties of the first radioligand that binds exclusively to the ECD of the CRF1 receptor. This ligand, which we named [¹²5I]Yamada peptide 20 ([¹²5I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004). We confirmed its high affinity for the [¹²5I]CRF binding site on the hCRF1 receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF1-CHO cells. Under optimized conditions, 20 pM [¹²5I]YP20 reproducibly bound to hCRF1-CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF1 receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated K(d) of ≈0.9 nM. The kinetic association of 20 pM [¹²5I]YP20 binding best fit to a rapid component (t(1/2)=0.69 min) and a sluggish component (t(1/2)=42 min). [¹²5I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t(1/2)=0.92 min and t(1/2)=11.7 min). While [¹²5I]YP20's binding kinetics are complex, its high affinity and pharmacological specificity indicate that it is an excellent radioligand for probing the ECD site of the CRF1 receptor.
Assuntos
Hormônio Liberador da Corticotropina/antagonistas & inibidores , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Células CHO , Hormônio Liberador da Corticotropina/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Guanilil Imidodifosfato/metabolismo , Humanos , Radioisótopos do Iodo , Cinética , Ligantes , Peptídeos/antagonistas & inibidores , Pirimidinas/metabolismo , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Receptores de Hormônio Liberador da Corticotropina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismoRESUMO
Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the (31)P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas rasRESUMO
Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac.RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac.RhoGDI complex dissociation ( Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem. 281, 19204-19219 ). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of "resting cell membrane" composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP).RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P(3) in the liposomes. Dissociation of Rac1(GDP).RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P(3) and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P(3) responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP).RhoGDI complexes, p67(phox), GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P(3) at a level superior to that of native membranes.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Lipossomos/metabolismo , Neuropeptídeos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Ativação Enzimática , Guanilil Imidodifosfato/metabolismo , Cobaias , Camundongos , Complexos Multiproteicos/metabolismo , NADPH Oxidases/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteínas rac1 de Ligação ao GTP , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Secretory lysosomes constitute a heterogeneous organelle of hematopoietic cells that combines the properties of regular lysosomes with those of secretory granules. Although secretory lysosomes serve essential functions, such as in the immune system and blood clotting, the mechanisms underlying the release of contents are incompletely understood. It is clear, however, that rab27a and the C2 domain protein munc13-4 serve essential functions. Mutations in these genes lead to immune disorders where the lytic granule function of cytotoxic T cells is jeopardized in humans. We identified munc13-4 as a rab27a binding protein from spleen. Munc13-4 is highly expressed in several hematopoietic cells including cytotoxic T cells and mast cells. We describe the molecular features of the interaction and requirements for localization, and show that munc13-4 is a positive regulator of secretory lysosome exocytosis.
Assuntos
Proteínas de Membrana/análise , Proteínas rab de Ligação ao GTP/análise , Animais , Citosol/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Baço/citologia , Baço/fisiologia , Sus scrofa , Transfecção/métodos , Proteínas rab27 de Ligação ao GTPRESUMO
Polypeptides of approximately 39, 36 and Assuntos
Células Fotorreceptoras Retinianas Bastonetes/química
, Rodopsina/química
, Roedores
, Transducina/química
, Transducina/isolamento & purificação
, Animais
, Bovinos
, Guanosina Trifosfato/análogos & derivados
, Guanosina Trifosfato/química
, Guanosina Trifosfato/metabolismo
, Guanilil Imidodifosfato/química
, Guanilil Imidodifosfato/metabolismo
, Luz
, Subunidades Proteicas/química
, Subunidades Proteicas/isolamento & purificação
, Subunidades Proteicas/metabolismo
, Células Fotorreceptoras Retinianas Bastonetes/metabolismo
, Rodopsina/metabolismo
, Roedores/metabolismo
, Transducina/metabolismo
RESUMO
RhoC is a member of the Rho family of Ras-related (small) GTPases and shares significant sequence similarity with the founding member of the family, RhoA. However, despite their similarity, RhoA and RhoC exhibit different binding preferences for effector proteins and give rise to distinct cellular outcomes, with RhoC being directly implicated in the invasiveness of cancer cells and the development of metastasis. While the structural analyses of the signaling-active and -inactive states of RhoA have been performed, thus far, the work on RhoC has been limited to an X-ray structure for its complex with the effector protein, mDia1 (for mammalian Diaphanous 1). Therefore, in order to gain insights into the molecular basis for RhoC activation, as well as clues regarding how it mediates distinct cellular responses relative to those induced by RhoA, we have undertaken a structural comparison of RhoC in its GDP-bound (signaling-inactive) state versus its GTP-bound (signaling-active) state as induced by the nonhydrolyzable GTP analogues, guanosine 5'-(beta,gamma-iminotriphosphate) (GppNHp) and guanosine 5'-(3-O-thiotriphosphate) (GTPgammaS). Interestingly, we find that GppNHp-bound RhoC only shows differences in its switch II domain, relative to GDP-bound RhoC, whereas GTPgammaS-bound RhoC exhibits differences in both its switch I and switch II domains. Given that each of the nonhydrolyzable GTP analogues is able to promote the binding of RhoC to effector proteins, these results suggest that RhoC can undergo at least two conformational transitions during its conversion from a signaling-inactive to a signaling-active state, similar to what has recently been proposed for the H-Ras and M-Ras proteins. In contrast, the available X-ray structures for RhoA suggest that it undergoes only a single conformational transition to a signaling-active state. These and other differences regarding the changes in the switch domains accompanying the activation of RhoA and RhoC provide plausible explanations for the functional specificity exhibited by the two GTPases.
Assuntos
Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Simulação por Computador , Cristalização , Cristalografia por Raios X , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/química , Guanilil Imidodifosfato/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais , Proteínas rho de Ligação ao GTP/isolamento & purificação , Proteína de Ligação a GTP rhoCRESUMO
The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a single-pass transmembrane glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. However, its role in signal transduction after IGF-II binding remains unclear. In the present study, we report that IGF-II/M6P receptor in the rat brain is coupled to a G-protein and that its activation by Leu27IGF-II, an analog that binds rather selectively to the IGF-II/M6P receptor, potentiates endogenous acetylcholine release from the rat hippocampal formation. This effect is mediated by a pertussis toxin (PTX)-sensitive GTP-binding protein and is dependent on protein kinase Calpha (PKCalpha)-induced phosphorylation of downstream substrates, myristoylated alanine-rich C kinase substrate, and growth associated protein-43. Additionally, treatment with Leu27IGF-II causes a reduction in whole-cell currents and depolarization of cholinergic basal forebrain neurons. This effect, which is blocked by an antibody against the IGF-II/M6P receptor, is also sensitive to PTX and is mediated via activation of a PKC-dependent pathway. These results together revealed for the first time that the single transmembrane domain IGF-II/M6P receptor expressed in the brain is G-protein coupled and is involved in the regulation of central cholinergic function via the activation of specific intracellular signaling cascades.
Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Proteína Quinase C-alfa/fisiologia , Receptor IGF Tipo 2/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Substituição de Aminoácidos , Animais , Ligação Competitiva , Toxina da Cólera/farmacologia , Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Canais de Potássio de Retificação Tardia/fisiologia , Proteína GAP-43/fisiologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanilil Imidodifosfato/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/análogos & derivados , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Isoproterenol/farmacologia , Masculino , Proteínas de Membrana/fisiologia , Substrato Quinase C Rico em Alanina Miristoilada , Proteínas do Tecido Nervoso/imunologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Toxina Pertussis/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 2/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The guanine nucleotide binding protein Ras plays a central role as molecular switch in cellular signal transduction. Ras cycles between a GDP-bound "off" state and a GTP-bound "on" state. Specific oncogenic mutations in the Ras protein are found in up to 30% of all human tumors. Previous 31P NMR studies had demonstrated that in liquid solution different conformational states in the GDP-bound as well as in the GTP-bound form coexist. High-field EPR spectroscopy of the GDP complexes in solution displayed differences in the ligand sphere of the wild-type complex as compared to its oncogenic mutant Ras(G12V). Only three water ligands were found in the former with respect to four in the G12V mutant [Rohrer, M. et al. (2001) Biochemistry 40, 1884-1889]. These differences were not detected in previous X-ray structures in the crystalline state. In this paper, we employ high-frequency electron nuclear double resonance (ENDOR) spectroscopy to probe the ligand sphere of the metal ion in the GDP-bound state. This technique in combination with selective isotope labeling has enabled us to detect the resonances of nuclei in the first ligand sphere of the ion with high spectral resolution. We have observed the 17O ENDOR spectra of the water ligands, and we have accurately determined the 17O hyperfine coupling with a(iso) = -0.276 mT, supporting the results of previous line shape analysis in solution. Further, the distinct resonances of the alpha-, beta-, and gamma-phosphorus of the bound nucleotides are illustrated in the 31P ENDOR spectra, and their hyperfine tensors lead to distances in agreement with the X-ray structures. Finally, 13C ENDOR spectra of uniformly 13C-labeled Ras(wt) x GDP and Ras(G12V) x GDP complexes as well as of the Ras(wt) x GppNHp and the selectively 1,4-13C-Asp labeled Ras(wt) x GDP complexes have revealed that in frozen solution only one amino acid is ligated to the ion in the GDP state, whereas two are bound in the GppNHp complex. Our results suggest that a second conformational state of the protein, if correlated with a different ligand sphere of the Mn2+ ion, is not populated in the GDP form of Ras at low temperatures in frozen solution.
Assuntos
Guanosina Difosfato/química , Metais/química , Oncogenes , Proteínas ras/química , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Congelamento , Glicina/genética , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/química , Guanilil Imidodifosfato/metabolismo , Humanos , Marcação por Isótopo , Metais/metabolismo , Mutação , Nucleotídeos/química , Nucleotídeos/metabolismo , Fósforo/química , Fósforo/metabolismo , Soluções/química , Valina/genética , Água/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Although some members of Ras family small GTPases, including M-Ras, share the primary structure of their effector regions with Ras, they exhibit vastly different binding properties to Ras effectors such as c-Raf-1. We have solved the crystal structure of M-Ras in the GDP-bound and guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p)-bound forms. The overall structure of M-Ras resembles those of H-Ras and Rap2A, except that M-Ras-Gpp(NH)p exhibits a distinctive switch I conformation, which is caused by impaired intramolecular interactions between Thr-45 (corresponding to Thr-35 of H-Ras) of the effector region and the gamma-phosphate of Gpp(NH)p. Previous 31P NMR studies showed that H-Ras-Gpp(NH)p exists in two interconverting conformations, states 1 and 2. Whereas state 2 is a predominant form of H-Ras and corresponds to the "on" conformation found in the complex with effectors, state 1 is thought to represent the "off" conformation, whose tertiary structure remains unknown. 31P NMR analysis shows that free M-Ras-Gpp(NH)p predominantly assumes the state 1 conformation, which undergoes conformational transition to state 2 upon association with c-Raf-1. These results indicate that the solved structure of M-Ras-Gp-p(NH)p corresponds to the state 1 conformation. The predominance of state 1 in M-Ras is likely to account for its weak binding ability to the Ras effectors, suggesting the importance of the tertiary structure factor in small GTPase-effector interaction. Further, the first determination of the state 1 structure provides a molecular basis for developing novel anti-cancer drugs as compounds that hold Ras in the state 1 "off" conformation.
Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Estrutura Terciária de Proteína , Proteínas ras/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/química , Guanilil Imidodifosfato/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Alinhamento de Sequência , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.
Assuntos
Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Guanilil Imidodifosfato/química , Proteínas ras/química , Substituição de Aminoácidos/genética , Cátions Bivalentes/química , Medição da Troca de Deutério , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Cinética , Magnésio/química , Espectroscopia de Ressonância Magnética/métodos , Fosfatos/química , Fosfatos/metabolismo , Isótopos de Fósforo/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Termodinâmica , Fator ral de Troca do Nucleotídeo Guanina/química , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, Talpha and Tbetagamma. N,N'-1,2-phenylenedimaleimide (o-PDM) and N,N'-1,4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of Talpha with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and >200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of Tbetagamma with both reagents produced a major species of 46-kDa. The combination of intact Talpha and o-PDM- or p-PDM-treated Tbetagamma reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified Talpha was incubated with intact Tbetagamma, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on Talpha represent functionally important residues of T.
Assuntos
Reagentes de Ligações Cruzadas/química , Cisteína/química , Transducina/química , Animais , Bovinos , Cisteína/metabolismo , Guanilil Imidodifosfato/metabolismo , Fenantrolinas/química , Fenilenodiaminas/química , Subunidades Proteicas , Segmento Externo da Célula Bastonete/química , Fatores de Tempo , Transducina/metabolismoRESUMO
We have examined the cellular processes underlying the desensitization of the 5-hydroxytryptamine (5-HT)(2A) receptor induced by agonist or antagonist exposure. Treatment of C6 glioma cells with either 5-HT or the 5-HT(2A) receptor antagonist ketanserin resulted in an attenuation in 5-HT(2A) receptor function, specifically the accumulation of inositol phosphates stimulated by the partial agonist quipazine. 5-HT-induced desensitization of the 5-HT(2A) receptor involved receptor internalization through a clathrin- and dynamin-dependent process because it was prevented by concanavalin A, monodansylcadaverine, and by expression of the dominant negative mutants beta-arrestin (319-418) and dynamin K44A. Although short-term (i.e., 10 min) 5-HT and ketanserin exposure resulted in the same degree of desensitization, ketanserin-induced desensitization was not prevented by these agents and did not involve receptor internalization. In contrast, prolonged ketanserin exposure (i.e., 2 h) resulted in 5-HT(2A) receptor internalization through a clathrin- and dynamin-dependent process, as was observed after agonist treatment. Inhibitors of protein kinase C or calcium-calmodulin kinase II did not attenuate or prevent 5-HT-induced desensitization of the receptor. 5-HT(2A) receptor desensitization induced by 5-HT and prolonged ketanserin treatment, but not by short-term ketanserin treatment, was prevented by the expression of the dominant negative mutant of G protein-coupled receptor kinase (GRK)2, GRK2-K220R, and by an anti-GRK2/3 antibody. Our data indicate a dual mechanism of early and late desensitization by the antagonist ketanserin. Short-term ketanserin treatment reduced the specific binding of the agonist radioligand [(125)I](+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([(125)I]DOI) and the ability of 5'-guanylylimidodiphosphate to attenuate this binding, suggesting that at the early stage of antagonist-induced desensitization the capacity of the 5-HT(2A) receptor to couple to G protein is impaired.
Assuntos
Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Arrestina/genética , Arrestina/metabolismo , Clatrina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas , Ensaio de Imunoadsorção Enzimática , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Hidrólise , Inositol/metabolismo , Ketanserina/metabolismo , Ketanserina/farmacologia , Ligantes , Receptor 5-HT2A de Serotonina , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/metabolismo , Transfecção , Células Tumorais Cultivadas , Quinases de Receptores Adrenérgicos betaRESUMO
Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.
Assuntos
Genes ras/genética , Melanoma/genética , Mutação Puntual , Códon , DNA de Neoplasias/genética , Éxons , Feminino , Guanilil Imidodifosfato/metabolismo , Humanos , Masculino , Melanoma/patologia , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , Estudos Retrospectivos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.
Assuntos
Nucleotídeos de Guanina/metabolismo , Rodopsina/metabolismo , Compostos de Sulfidrila/química , Transducina/química , Transducina/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Bovinos , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Nucleotídeos de Guanina/química , Guanilil Imidodifosfato/metabolismo , Luz , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Substâncias Redutoras/química , Substâncias Redutoras/farmacologia , Rodopsina/farmacologia , Rodopsina/efeitos da radiação , Segmento Externo da Célula Bastonete/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estilbenos/química , Estilbenos/farmacologia , Reagentes de Sulfidrila/química , Reagentes de Sulfidrila/farmacologia , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Tiocianatos/química , Tiocianatos/farmacologia , Transducina/efeitos dos fármacosRESUMO
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.