RESUMO
Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula Theraphosa apophysis that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized NaV-targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for NaVs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target NaV1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of NaV channels observed in vitro translated in vivo, with reversal of nocifensive behaviours in a murine model of NaV-mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the NaV channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed in vivo. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting NaV channels, and the research into novel drugs to treat neurological diseases.
RESUMO
Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.
Assuntos
Venenos Elapídicos/enzimologia , Elapidae/classificação , Elapidae/genética , Evolução Molecular , Fosfolipases A2 do Grupo IV/genética , Dor , Células Receptoras Sensoriais/fisiologia , Adaptação Biológica/genética , Animais , Venenos Elapídicos/genética , Filogenia , Células Receptoras Sensoriais/metabolismoRESUMO
Vincristine is an antineoplastic substance that is part of many chemotherapy regimens, used especially for the treatment of a variety of pediatric cancers including leukemias and brain tumors. Unfortunately, many vincristine-treated patients develop peripheral neuropathy, a side effect characterized by sensory, motoric, and autonomic symptoms. The sensory symptoms include pain, in particular hypersensitivity to light touch, as well as loss of sensory discrimination to detect vibration and touch. The symptoms of vincristine-induced neuropathy are only poorly controlled by currently available analgesics and therefore often necessitate dose reductions or even cessation of treatment. The aim of this study was to identify new therapeutic targets for the treatment of vincristine-induced peripheral neuropathy (VIPN) by combining behavioral experiments, histology, and pharmacology after vincristine treatment. Local intraplantar injection of vincristine into the hind paw caused dose- and time-dependent mechanical hypersensitivity that developed into mechanical hyposensitivity at high doses, and lead to a pronounced, dose-dependent infiltration of immune cells at the site of injection. Importantly, administration of minocycline effectively prevented the development of mechanical hypersensitivity and infiltration of immune cells in mouse models of vincristine induce peripheral neuropathy (VIPN) based on intraperitoneal or intraplantar administration of vincristine. Similarly, Toll-like receptor 4 knockout mice showed diminished vincristine-induced mechanical hypersensitivity and immune cell infiltration, while treatment with the anti-inflammatory meloxicam had no effect. These results provide evidence for the involvement of Toll-like receptor 4 in the development of VIPN and suggest that minocycline and/or direct Toll-like receptor 4 antagonists may be an effective preventative treatment for patients receiving vincristine.
RESUMO
Despite significant advances in our understanding of the molecular basis of pain, the precise contributions of individual genes to our perception of this primal sensation remains incomplete. However, transcriptomic studies - providing a snapshot of the mRNA expression of a given cell or tissue - have considerably increased insight into the gene expression fingerprint of specific sensory neuronal subtypes, as well as gene expression changes that occur in diverse pathologies associated with pain. Moreover, transcriptomic studies have accelerated the identification of venom-derived peptides that may provide novel leads for the development of analgesics. This review discusses some of the key techniques, insights and limitations of transcriptomic studies that have contributed to pain research and highlights how the application of transcriptomics can be used to accelerate analgesic venom peptide drug discovery.
Assuntos
Perfilação da Expressão Gênica , Dor/etiologia , Pesquisa , Transcriptoma , Animais , Biotecnologia/métodos , Biologia Computacional/métodos , Descoberta de Drogas , Perfilação da Expressão Gênica/métodos , Humanos , Especificidade de Órgãos/genética , Dor/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteômica/métodosRESUMO
Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels
Assuntos
Anestesiologia , ToxicologiaRESUMO
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
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
Mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin at effector region threonine 35 has diverse effects on the Ras GTPase cycle, the dominant one of which is the inhibition of Ras-Raf coupling, leading to complete blockade of Ras downstream signaling. To understand the structural basis of the functional consequences of glucosylation, the X-ray crystal structure of glucosylated Ras-GDP was compared with that of non-modified Ras. Glucosylated Ras exhibits a different crystal packing but the overall three-dimensional structure is not altered. The glucose group does not affect the conformation of the effector loop. Due to steric constraints, the glucose moiety prevents the formation of the GTP conformation of the effector loop, which is a prerequisite for binding to the Raf-kinase. The X-ray crystal data also revealed the alpha-anomeric configuration of the bound glucose, indicating that the glucose transfer proceeds under retention of the C-1 configuration of the d-alpha-glucose. Therefore, glucosylation preserves the inactive conformation of the effector loop independently of the nucleotide occupancy, leading to a complete inhibition of downstream signaling of Ras.
Assuntos
Toxinas Bacterianas/metabolismo , Clostridium , Citotoxinas/metabolismo , Glucose/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Toxinas Bacterianas/toxicidade , Cristalografia por Raios X , Citotoxinas/toxicidade , Guanosina Difosfato/metabolismo , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Treonina/metabolismo , Proteínas ras/antagonistas & inibidoresRESUMO
Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.
Assuntos
Bacteriófago T4/enzimologia , Muramidase/química , Polímeros/química , Dobramento de Proteína , Cisteína/química , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Guanidina/farmacologia , Microscopia de Força Atômica , Modelos Moleculares , Dados de Sequência Molecular , Oxigênio/química , Estresse MecânicoRESUMO
The past three years have seen the solution of several nuclear transport component structures and recently of the structure of a regulator bound to part of a nuclear pore complex (NPC) protein. These structures have provided a wealth of valuable information about the proteins involved and suggested strategies for further investigation of their properties. We do not have space here to go into detail about this information, so instead we are illustrating the structures and providing primary references enabling interested readers to find further information. On this page, we are concentrating on the GTPase Ran and proteins that modulate its activity, and on the facing page are the other transport factors, some of which also interact directly with Ran. Notably absent at the moment are the nuclear pore complex component s, apart from one domain of RanBP2. Only when theses are characterized fully will we really be able to understand how transport substrates move across the nuclear envelope.
Assuntos
Núcleo Celular/fisiologia , Animais , Transporte Biológico , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
rna1p is the Schizosaccharomyces pombe ortholog of the mammalian GTPase-activating protein (GAP) of Ran. Both proteins are essential for nuclear transport. Here, we report the crystal structure of rna1p at 2.66 A resolution. It contains 11 leucine-rich repeats that adopt the nonglobular shape of a crescent, bearing no resemblance to RhoGAP or RasGAP. The invariant residues of RanGAP form a contiguous surface, strongly indicating the Ran-binding interface. Alanine mutations identify Arg-74 as a critical residue for GTP hydrolysis. In contrast to RasGAP and RhoGAP, Arg-74 could be substituted by lysine and contributed significantly to the binding of Ran. Therefore, we suggest a GAP mechanism for rna1p, which constitutes a variation of the arginine finger mechanism found for Ras GAP and RhoGAP.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas/química , Proteínas/metabolismo , Schizosaccharomyces/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Leucina/química , Leucina/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Proteína ran de Ligação ao GTP , Proteínas Ativadoras de ras GTPaseRESUMO
We identified a new, unique upstream activating sequence (5'-GGTGGCAAA-3') in the promoters of 26 out of the 32 proteasomal yeast genes characterized to date, which we propose to call proteasome-associated control element. By using the one-hybrid method, we show that the factor binding to the proteasome-associated control element is Rpn4p, a protein containing a C2H2-type finger motif and two acidic domains. Electrophoretic mobility shift assays using proteasome-associated control element sequences from two regulatory proteasomal genes confirmed specific binding of purified Rpn4p to these sequences. The role of Rpn4p to function as a transregulator in yeast is corroborated by its ability of stimulating proteasome-associated control element-driven lacZ expression and by experiments using the RPT4 and RPT6 gene promoters coupled to the bacterial cat gene as a reporter. Additionally, we found the proteasome-associated control element to occur in a number of promoters to genes which are related to the ubiquitin-proteasome pathway in yeast.
Assuntos
Cisteína Endopeptidases/genética , Proteínas de Ligação a DNA/genética , Complexos Multienzimáticos/genética , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Leveduras/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA/química , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Reporter , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Complexo de Endopeptidases do Proteassoma , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/química , Dedos de Zinco/genéticaRESUMO
The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase , Proteínas ras/metabolismo , Animais , Sequência Conservada , Proteínas de Ligação ao GTP/genética , Nucleotídeos de Guanina/metabolismo , Guanilil Imidodifosfato/metabolismo , Haplorrinos , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas ral de Ligação ao GTP , Proteínas ras/genéticaRESUMO
Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.
Assuntos
Proteínas Nucleares/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Cristalografia por Raios X , Drosophila , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Carioferinas , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oryza , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína ran de Ligação ao GTPRESUMO
The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas ras/química , Cristalografia por Raios X , Proteínas de Ligação ao GTP/fisiologia , Produtos do Gene vpr/química , Produtos do Gene vpr/fisiologia , Modelos Moleculares , Mutagênese , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais , Fator ral de Troca do Nucleotídeo Guanina , Proteínas rap de Ligação ao GTP , Proteínas ras/fisiologiaRESUMO
The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação ao GTP/genética , Espectrometria de Fluorescência/métodos , Difração de Raios X/métodos , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação ao GTP/química , Guanilil Imidodifosfato/química , Humanos , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Placenta/química , Ligação Proteica , Fatores de Tempo , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTP , Proteínas ras/química , Proteína rhoA de Ligação ao GTPRESUMO
RCC1, the regulator of chromosome condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, alpha = 113.0, beta = 103.9,gamma = 103.3 degrees. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Humanos , Proteínas Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.