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1.
Plant Physiol ; 182(4): 2143-2153, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32015077

RESUMO

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K+). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K+ fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K+ channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein-protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K+ transport.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Canais de Potássio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Mutação Puntual , Potássio/metabolismo , Canais de Potássio/genética , Proteínas Serina-Treonina Quinases/genética
2.
Proc Natl Acad Sci U S A ; 114(6): E999-E1008, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28119500

RESUMO

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.


Assuntos
Proteínas de Drosophila/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Fenotiazinas/farmacologia , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Antipsicóticos/química , Antipsicóticos/farmacologia , Cristalografia por Raios X , Modelos Animais de Doenças , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Síndrome do Cromossomo X Frágil/genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas Sensoras de Cálcio Neuronal/química , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/química , Neuropeptídeos/genética , Fenotiazinas/química , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Sinapses/genética
3.
Proc Natl Acad Sci U S A ; 113(3): E396-405, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26719420

RESUMO

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Transdução de Sinais , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/química , Sítios de Ligação , Calorimetria , Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Modelos Biológicos , Fenótipo , Fosfolipídeos/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Soluções , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo
4.
Proc Natl Acad Sci U S A ; 111(42): E4532-41, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25288725

RESUMO

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Homeostase , Proteínas Serina-Treonina Quinases/química , Estresse Fisiológico , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Proteínas Quinases Dependentes de AMP Cíclico/química , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Transporte de Íons , Lítio/química , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Sódio/química
5.
J Cell Sci ; 127(Pt 19): 4246-59, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25074811

RESUMO

The conserved Ca(2+)-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gαs regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Cristalografia por Raios X/métodos , Proteínas de Drosophila/metabolismo , Humanos , Junção Neuromuscular/metabolismo , Transmissão Sináptica
6.
Mol Cell ; 30(4): 507-18, 2008 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-18498752

RESUMO

Pygo and BCL9/Legless transduce the Wnt signal by promoting the transcriptional activity of beta-catenin/Armadillo in normal and malignant cells. We show that human and Drosophila Pygo PHD fingers associate with their cognate HD1 domains from BCL9/Legless to bind specifically to the histone H3 tail methylated at lysine 4 (H3K4me). The crystal structures of ternary complexes between PHD, HD1, and two different H3K4me peptides reveal a unique mode of histone tail recognition: efficient histone binding requires HD1 association, and the PHD-HD1 complex binds preferentially to H3K4me2 while displaying insensitivity to methylation of H3R2. Therefore, this is a prime example of histone tail binding by a PHD finger (of Pygo) being modulated by a cofactor (BCL9/Legless). Rescue experiments in Drosophila indicate that Wnt signaling outputs depend on histone decoding. The specificity of this process provided by the Pygo-BCL9/Legless complex suggests that this complex facilitates an early step in the transition from gene silence to Wnt-induced transcription.


Assuntos
Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/química , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Fatores de Transcrição
7.
J Struct Biol ; 190(2): 162-72, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25816760

RESUMO

The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Bacteriocinas/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Antibacterianos/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Cristalização , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Oligonucleotídeos/genética , Conformação Proteica , Engenharia de Proteínas/métodos , Eletricidade Estática , Ultracentrifugação
8.
Int J Mol Sci ; 14(3): 5734-49, 2013 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-23481636

RESUMO

The Arabidopsis SOS2 family of twenty-six protein kinases (CIPKs), their interacting activators, the SOS3 family of ten calcium-binding proteins (CBLs) and protein phosphatases type 2C (PP2C), function together in decoding calcium signals elicited by different environmental stimuli. Biochemical data suggest that stable CBL-CIPK or CIPK-PP2C complexes may be regulating the activity of various substrates controlling ion homeostasis. The available structural information provides a general regulatory mechanism in which calcium perception by CBLs and kinase activation is coupled. The structural basis of this molecular mechanism and the specificity of the network is reviewed and discussed in detail.

9.
Elife ; 122023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38018500

RESUMO

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.


Assuntos
Cálcio , Fatores de Troca do Nucleotídeo Guanina , Cálcio/metabolismo , Fosforilação , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais , Chaperonas Moleculares/metabolismo
10.
Front Neurosci ; 16: 1007531, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36466176

RESUMO

Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.

11.
Nat Commun ; 10(1): 2798, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243268

RESUMO

Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.


Assuntos
Cálcio/metabolismo , Biblioteca Gênica , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Animais , Catálise , Células Cultivadas , Técnicas de Química Combinatória , Drosophila/fisiologia , Imageamento por Ressonância Magnética , Masculino , Membranas Artificiais , Camundongos , Proteínas Sensoras de Cálcio Neuronal/genética , Neurônios/metabolismo , Palmitoil-CoA Hidrolase , Permeabilidade , Conformação Proteica , Proteínas
12.
J Med Chem ; 61(14): 5910-5921, 2018 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-29966094

RESUMO

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Proteínas Sensoras de Cálcio Neuronal/antagonistas & inibidores , Neuropeptídeos/antagonistas & inibidores , Fenotiazinas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteínas Sensoras de Cálcio Neuronal/química , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Conformação Proteica em alfa-Hélice
13.
Artigo em Inglês | MEDLINE | ID: mdl-17620712

RESUMO

The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 A.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Arabidopsis/genética , Proteínas Serina-Treonina Quinases/química , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
14.
J Mol Biol ; 345(5): 1253-64, 2005 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-15644219

RESUMO

The Arabidopsis thaliana SOS3 gene encodes a calcium sensor that is required for plant salt tolerance. The SOS3 protein binds to and activates the self-inhibited SOS2 protein kinase, which mediates the expression and activities of various transporters important for ion homeostasis under salt stress. SOS3 belongs to a unique family of calcium-binding proteins that contain two pairs of EF hand motifs with four putative metal-binding sites. We report the crystal structure of a dimeric SOS3 protein in complex with calcium, and with calcium and manganese. Analytical ultracentrifugation experiments and circular dichroism measurements show that calcium binding is responsible for the dimerization of SOS3. This leads to a change in the global shape and surface properties of the protein that may be sufficient to transmit the Ca(2+) signal elicited during salt stress.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Cálcio/farmacologia , Doenças das Plantas/induzido quimicamente , Sais/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/química , Dicroísmo Circular , Cristalografia por Raios X , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Soluções
15.
Curr Protein Pept Sci ; 5(5): 399-416, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15544535

RESUMO

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Animais , Antibacterianos/imunologia , Antibacterianos/isolamento & purificação , Bacteriocinas/imunologia , Bacteriocinas/isolamento & purificação , Modelos Moleculares , Peptídeos Cíclicos/imunologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
16.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 4): 509-12, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24699751

RESUMO

The Arabidopsis thaliana K(+) transporter 1 (AKT1) participates in the maintenance of an adequate cell potassium (K(+)) concentration. The CBL-interacting protein kinase 23 (CIPK23) activates AKT1 for K(+) uptake under low-K(+) conditions. This process is mediated by the interaction between the cytosolic ankyrin-repeat (AR) domain of AKT1 and the kinase domain of CIPK23. However, the precise boundaries of the AR domain and the residues responsible for the interaction are still unknown. Here, the optimization procedure to obtain an AR domain construct suitable for crystallization and the preliminary crystallographic analysis of the obtained crystals are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 34.83, b = 65.89, c = 85.44 Å, and diffracted to 1.98 Šresolution.


Assuntos
Repetição de Anquirina , Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Cristalografia por Raios X/métodos , Canais de Potássio/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalização , Canais de Potássio/genética , Canais de Potássio/metabolismo
17.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 4): 530-4, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24699756

RESUMO

Drosophila melanogaster contains two calcium-binding proteins, Frq1 and Frq2, in the nervous system that control the number of synapses and the probability of release. To understand the differential function of the two proteins, whose sequence is only 5% dissimilar, the crystal structures of Frq1 and Frq2 are needed. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of Frq2 are presented. The full-length protein was purified using a two-step chromatographic procedure. Two different diffracting crystal forms were obtained using a progressive streak-seeding method and detergents.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/isolamento & purificação , Clonagem Molecular , Cristalização/métodos , Cristalografia por Raios X/métodos , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Drosophila/genética
18.
Structure ; 21(12): 2208-20, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24183574

RESUMO

Pygo proteins promote Armadillo- and ß-catenin-dependent transcription, by relieving Groucho-dependent repression of Wnt targets. Their PHD fingers bind histone H3 tail methylated at lysine 4, and to the HD1 domain of their Legless/BCL9 cofactors, linking Pygo to Armadillo/ß-catenin. Intriguingly, fly Pygo orthologs exhibit a tryptophan > phenylalanine substitution in their histone pocket-divider which reduces their affinity for histones. Here, we use X-ray crystallography and NMR, to discover a conspicuous groove bordering this phenylalanine in the Drosophila PHD-HD1 complex--a semi-aromatic cage recognizing asymmetrically methylated arginine 2 (R2me2a), a chromatin mark of silenced genes. Our structural model of the ternary complex reveals a distinct mode of dimethylarginine recognition, involving a polar interaction between R2me2a and its groove, the structural integrity of which is crucial for normal tissue patterning. Notably, humanized fly Pygo derepresses Notch targets, implying an inherent Notch-related function of classical Pygo orthologs, disabled in fly Pygo, which thus appears dedicated to Wnt signaling.


Assuntos
Arginina/análogos & derivados , Proteínas de Drosophila/química , Drosophila/metabolismo , Histonas/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Arginina/química , Cristalografia por Raios X , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolução Molecular , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo
19.
PLoS One ; 7(12): e52401, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23285027

RESUMO

Cell motility, adhesion and phagocytosis are controlled by actin and membrane remodelling processes. Bridging integrator-2 (Bin2) also called Breast cancer-associated protein 1 (BRAP1) is a predicted N-BAR domain containing protein with unknown function that is highly expressed in leucocytic cells. In the present study we solved the structure of Bin2 BAR domain and studied its membrane binding and bending properties in vitro and in vivo. Live-cell imaging experiments showed that Bin2 is associated with actin rich structures on the plasma membrane, where it was targeted through its N-BAR domain. Pull-down experiments and immunoprecipitations showed that Bin2 C-terminus bound SH3 domain containing proteins such as Endophilin A2 and α-PIX. siRNA of endogenous protein led to decreased cell migration, increased phagocytosis and reduced podosome density and dynamics. In contrast, overexpression of Bin2 led to decreased phagocytosis and increased podosome density and dynamics. We conclude that Bin2 is a membrane-sculpting protein that influences podosome formation, motility and phagocytosis in leucocytes. Further understanding of this protein may be key to understand the behaviour of leucocytes under physiological and pathological conditions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Extensões da Superfície Celular/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Fagocitose , Sequência de Aminoácidos , Animais , Adesão Celular , Movimento Celular , Cristalografia por Raios X , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana/química , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Ratos , Domínios de Homologia de src
20.
J Mol Biol ; 424(5): 283-94, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23022605

RESUMO

The Arabidopsisthaliana Na(+)/H(+) antiporter salt-overly-sensitive 1 (SOS1) is essential to maintain low intracellular levels of toxic Na(+) under salt stress. Available data show that the plant SOS2 protein kinase and its interacting activator, the SOS3 calcium-binding protein, function together in decoding calcium signals elicited by salt stress and regulating the phosphorylation state and the activity of SOS1. Molecular genetic studies have shown that the activation implies a domain reorganization of the antiporter cytosolic moiety, indicating that there is a clear relationship between function and molecular structure of the antiporter. To provide information on this issue, we have carried out in vivo and in vitro studies on the oligomerization state of SOS1. In addition, we have performed electron microscopy and single-particle reconstruction of negatively stained full-length and active SOS1. Our studies show that the protein is a homodimer that contains a membrane domain similar to that found in other antiporters of the family and an elongated, large, and structured cytosolic domain. Both the transmembrane (TM) and cytosolic moieties contribute to the dimerization of the antiporter. The close contacts between the TM and the cytosolic domains provide a link between regulation and transport activity of the antiporter.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Trocadores de Sódio-Hidrogênio/química , Proteínas de Arabidopsis/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Trocadores de Sódio-Hidrogênio/ultraestrutura
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