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1.
J Immunol ; 204(8): 2269-2276, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32198144

RESUMO

The B cell adaptor protein (BCAP) is a multimodular regulator of inflammatory signaling in diverse immune system cells. BCAP couples TLR signaling to phosphoinositide metabolism and inhibits MyD88-directed signal transduction. BCAP is recruited to the TLR signalosome forming multitypic interactions with the MAL and MyD88 signaling adaptors. In this study, we show that indirect dimerization of BCAP TIR is required for negative regulation of TLR signaling. This regulation is mediated by a transcription factor Ig (TIG/IPT) domain, a fold found in the NF-κB family of transcription factors. We have solved the crystal structure of the BCAP TIG and find that it is most similar to that of early B cell factor 1 (EBF1). In both cases, the dimer is stabilized by a helix-loop-helix motif at the C terminus and interactions between the ß-sheets of the Ig domains. BCAP is exclusively localized in the cytosol and is unable to bind DNA. Thus, the TIG domain is a promiscuous dimerization module that has been appropriated for a range of regulatory functions in gene expression and signal transduction.

2.
Structure ; 28(3): 281-289.e3, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-31995744

RESUMO

Toll-like receptors (TLRs) are pivotal in triggering the innate immune response to pathogen infection. Ligand binding induces receptor dimerization which facilitates the recruitment of other post-receptor signal transducers into a complex signalosome, the Myddosome. Central to this process is Myeloid differentiation primary response 88 (MyD88), which is required by almost all TLRs, and signaling is thought to proceed via the stepwise, sequential assembly of individual components. Here, we show that the death domains of human MyD88 spontaneously and reversibly associate to form helical filaments in vitro. A 3.1-Å cryoelectron microscopy structure reveals that the architecture of the filament is identical to that of the 6:4 MyD88-IRAK4-IRAK2 hetero-oligomeric Myddosome. Additionally, the death domain of IRAK4 interacts with the filaments to reconstitute the non-stoichiometric 6:4 MyD88-IRAK4 complex. Together, these data suggest that intracellularly, the MyD88 scaffold may be pre-formed and poised for recruitment of IRAKs on receptor activation and TIR engagement.

3.
Nucleic Acids Res ; 45(18): 10845-10860, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977623

RESUMO

In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação a RNA/química , Amino Açúcares/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
4.
J Biol Chem ; 292(2): 652-660, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27909057

RESUMO

Ligand binding to Toll-like receptors (TLRs) results in dimerization of their cytosolic Toll/interleukin-1 receptor (TIR) domains and recruitment of post-receptor signal transducers into a complex signalosome. TLR activation leads to the production of transcription factors and pro-inflammatory molecules and the activation of phosphoinositide 3-kinases (PI3K) in a process that requires the multimodular B-cell adaptor for phosphoinositide 3-kinase (BCAP). BCAP has a sequence previously proposed as a "cryptic" TIR domain. Here, we present the structure of the N-terminal region of human BCAP and show that it possesses a canonical TIR fold. Dimeric BCAP associates with the TIR domains of TLR2/4 and MAL/TIRAP, suggesting that it is recruited to the TLR signalosome by multitypic TIR-TIR interactions. BCAP also interacts with the p85 subunit of PI3K and phospholipase Cγ, enzymes that deplete plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), and these interactions provide a molecular explanation for BCAP-mediated down-regulation of inflammatory signaling.


Assuntos
Proteínas de Transporte/química , Multimerização Proteica , Transdução de Sinais , Receptor 2 Toll-Like/química , Receptor 4 Toll-Like/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/química , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fosfatidilinositol 3-Quinases , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Domínios Proteicos , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
5.
Int J Biol Macromol ; 82: 22-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26433176

RESUMO

Recent advances in glycobiology have revealed the essential role of lectins in deciphering the glycocodes at the cell surface to generate important biological signaling responses. ArtinM, a d-mannose-binding lectin isolated from the seeds of jackfruit (Artocarpus heterophyllus), is composed of 16 kDa subunits that are associated to form a homotetramer. Native ArtinM (n-ArtinM) exerts immunomodulatory and regenerative effects, but the potential pharmaceutical applicability of the lectin is highly limited by the fact that its production is expensive, laborious, and impossible to be scaled up. This led us to characterize a recombinant form of the lectin obtained by expression in Saccharomyces cerevisiae (y-ArtinM). In the present study, we demonstrated that y-ArtinM is similar to n-ArtinM in subunit arrangement, oligomerization and carbohydrate binding specificity. We showed that y-ArtinM can exert n-ArtinM biological activities such as erythrocyte agglutination, stimulation of neutrophil migration and degranulation, mast cell degranulation, and induction of interleukin-12 and interleukin-10 production by macrophages. In summary, the expression of ArtinM in yeast resulted in successful production of an active, recombinant form of ArtinM that is potentially useful for pharmaceutical application.


Assuntos
Carboidratos/química , Lectinas de Ligação a Manose/química , Estrutura Molecular , Proteínas Recombinantes , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Citocinas/biossíntese , Hemaglutinação , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Polissacarídeos/química , Ligação Proteica , Receptor 2 Toll-Like , Leveduras/genética , Leveduras/metabolismo
6.
J Mol Biol ; 415(1): 92-101, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22056329

RESUMO

In the biosynthesis of the clinically important antibiotic erythromycin D, the glycosyltransferase (GT) EryCIII, in concert with its partner EryCII, attaches a nucleotide-activated sugar to the macrolide scaffold with high specificity. To understand the role of EryCII, we have determined the crystal structure of the EryCIII·EryCII complex at 3.1 Å resolution. The structure reveals a heterotetramer with a distinctive, elongated quaternary organization. The EryCIII subunits form an extensive self-complementary dimer interface at the center of the complex, and the EryCII subunits lie on the periphery. EryCII binds in the vicinity of the putative macrolide binding site of EryCIII but does not make direct interactions with this site. Our biophysical and enzymatic data support a model in which EryCII stabilizes EryCIII and also functions as an allosteric activator of the GT.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Glicosiltransferases/biossíntese , Glicosiltransferases/química , Oxirredutases/biossíntese , Oxirredutases/química , Regulação Alostérica , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Eritromicina/biossíntese , Eritromicina/química , Eritromicina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade
7.
J Biol Chem ; 284(37): 25404-11, 2009 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-19592493

RESUMO

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/química , Fator 88 de Diferenciação Mieloide/química , Linhagem Celular , Reagentes para Ligações Cruzadas/farmacologia , Regulação da Expressão Gênica , Humanos , Espectrometria de Massas/métodos , Microdomínios da Membrana/química , Modelos Biológicos , Estrutura Terciária de Proteína , Espalhamento de Radiação , Transdução de Sinais , Ultracentrifugação , Raios X , Receptor fas/metabolismo
8.
Immunol Rev ; 227(1): 161-75, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19120483

RESUMO

Initiation of the innate immune response requires agonist recognition by a pathogen recognition receptor. Following ligand binding, conformational rearrangement of the receptor creates a molecular scaffold from which signal transduction is propagated via complex cellular signaling pathways. This in turn leads to the induction of a pro-inflammatory immune response. A critical component of these signaling pathways is the homotypic interaction of receptor and adapter proteins via specific protein interaction domains. Within the innate immune signaling cascade, homotypic interactions between members of the death domain family and the Toll/interleukin-1 receptor domain are particularly important. Here we discuss the current understanding of the molecular basis of these homotypic receptor:adapter interactions and their role in innate immune signal transduction.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas de Helminto/imunologia , Imunidade Inata , Receptores Citoplasmáticos e Nucleares/imunologia , Transdução de Sinais/imunologia , Regulação Alostérica/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/imunologia , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional/imunologia , Transporte Proteico/imunologia , Receptores Citoplasmáticos e Nucleares/química , Relação Estrutura-Atividade , Receptores Toll-Like/química , Receptores Toll-Like/imunologia
9.
J Biol Chem ; 283(48): 33447-54, 2008 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-18829464

RESUMO

The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Antígenos de Diferenciação/química , Proteínas de Drosophila/química , Modelos Biológicos , Complexos Multiproteicos/química , Proteínas Serina-Treonina Quinases/química , Receptores Imunológicos/química , Transdução de Sinais/fisiologia , Receptores Toll-Like/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
10.
FEBS J ; 274(9): 2196-209, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17419733

RESUMO

Domains within the multienzyme polyketide synthases are linked by noncatalytic sequences of variable length and unknown function. Recently, the crystal structure was reported of a portion of the linker between the acyltransferase (AT) and ketoreductase (KR) domains from module 1 of the erythromycin synthase (6-deoxyerythronolide B synthase), as a pseudodimer with the adjacent ketoreductase (KR). On the basis of this structure, the homologous linker region between the dehydratase (DH) and enoyl reductase (ER) domains in fully reducing modules has been proposed to occupy a position on the periphery of the polyketide synthases complex, as in porcine fatty acid synthase. We report here the expression and characterization of the same region of the 6-deoxyerythronolide B synthase module 1 AT-KR linker, without the adjacent KR domain (termed DeltaN AT1-KR1), as well as the corresponding section of the DH-ER linker. The linkers fold autonomously and are well structured. However, analytical gel filtration and ultracentrifugation analysis independently show that DeltaN AT1-KR1 is homodimeric in solution; site-directed mutagenesis further demonstrates that linker self-association is compatible with the formation of a linker-KR pseudodimer. Our data also strongly indicate that the DH-ER linker associates with the upstream DH domain. Both of these findings are incompatible with the proposed model for polyketide synthase architecture, suggesting that it is premature to allocate the linker regions to a position in the multienzymes based on the solved structure of animal fatty acid synthase.


Assuntos
Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Dobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Homologia Estrutural de Proteína , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Clonagem Molecular , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Policetídeo Sintases/genética , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
J Biol Chem ; 282(18): 13522-31, 2007 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-17324925

RESUMO

The cytokine Spätzle is the ligand for Drosophila Toll, the prototype of an important family of membrane receptors that function in embryonic patterning and innate immunity. A dimeric precursor of Spätzle is processed by an endoprotease to produce a form (C-106) that cross-links Toll receptor ectodomains and establishes signaling. Here we show that before processing the pro-domain of Spätzle is required for correct biosynthesis and secretion. We mapped two loss-of-function mutations of Spätzle to a discrete site in the pro-domain and showed that the phenotype arises because of a defect in biosynthesis rather than signaling. We also report that the pro-domain and C-106 remain associated after cleavage and that this processed complex signals with the same characteristics as the C-terminal fragment. These results suggest that before activation the determinants on C-106 that bind specifically to Toll are sequestered by the pro-domain and that proteolytic processing causes conformational rearrangements that expose these determinants and enables binding to Toll. Furthermore, we show that the pro-domain is released when the Toll extracellular domain binds to the complex, a finding that has implications for the generation of a signaling-competent Toll dimer.


Assuntos
Citocinas/biossíntese , Proteínas de Drosophila/biossíntese , Processamento de Proteína Pós-Traducional/fisiologia , Receptores Toll-Like/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Dimerização , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ligantes , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Transdução de Sinais/fisiologia
12.
FEBS Lett ; 579(18): 3920-6, 2005 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-16004997

RESUMO

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. We present the NMR solution structure of the isolated death domain of Pelle and a study of the interaction between the DDs of Pelle and Tube. Our data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Cristalografia por Raios X , Dimerização , Drosophila , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ultracentrifugação
13.
J Biol Chem ; 280(39): 33453-60, 2005 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-16009712

RESUMO

Nerve growth factor (NGF) is the ligand for two unrelated cellular receptors, TrkA and p75(NTR), and acts as a mediator in the development and maintenance of the mammalian nervous system. Signaling through TrkA kinase domains promotes neuronal survival, whereas activation of the p75(NTR) "death domains" induces apoptosis under correct physiological conditions. However, co-expression of these receptors leads to enhanced neuronal survival upon NGF stimulation, possibly through a ternary p75(NTR) x NGF x TrkA complex. We have expressed human p75(NTR) ligand binding domain as a secreted glycosylated protein in Trichoplusia ni cells. Following assembly and purification of soluble p75(NTR) x NGF complexes, mass spectrometry, analytical ultracentrifugation, and solution x-ray scattering measurements are indicative of 2:2 stoichiometry, which implies a symmetric complex. Molecular models of the 2:2 p75(NTR) x NGF complex based on these data are not consistent with the further assembly of either symmetric (2:2:2) or asymmetric (2:2:1) ternary p75(NTR) x NGF x TrkA complexes.


Assuntos
Fator de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/química , Receptor de Fator de Crescimento Neural/metabolismo , Cromatografia em Gel , Simulação por Computador , Cisteína/química , Humanos , Luz , Espectrometria de Massas , Modelos Moleculares , Peso Molecular , Fator de Crescimento Neural/química , Estrutura Terciária de Proteína , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/isolamento & purificação , Receptor trkA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ultracentrifugação
14.
J Magn Reson ; 175(2): 222-5, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15907387

RESUMO

Calibration of the 3J(NC(gamma)) couplings across the N-C(alpha)-C(beta)-C(gamma) fragment of aspartate and asparagine residues is afforded by two interactions that produce fixed conformations of the side chains in solution. One is the binding of these side chains to calcium ions; the other is the H-bond interaction of these side chains with a backbone amide.


Assuntos
Asparagina/química , Ácido Aspártico/química , Proteínas de Ligação ao Cálcio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Calibragem , Estrutura Molecular , Conformação Proteica
15.
J Biol Chem ; 280(24): 22793-9, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15795223

RESUMO

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spätzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spätzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Spätzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.


Assuntos
Proteínas de Drosophila/química , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Biofísica , Padronização Corporal , Calorimetria , Linhagem Celular , Reagentes para Ligações Cruzadas/farmacologia , Citocinas/metabolismo , Dimerização , Drosophila , Proteínas de Drosophila/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Ligantes , Luciferases/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Receptores Toll-Like , Ultracentrifugação
16.
J Mol Biol ; 341(3): 839-52, 2004 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-15288790

RESUMO

FtsZ is part of a mid-cell cytokinetic structure termed the Z-ring that recruits a hierarchy of fission related proteins early in the bacterial cell cycle. The widely conserved ZapA has been shown to interact with FtsZ, to drive its polymerisation and to promote FtsZ filament bundling thereby contributing to the spatio-temporal tuning of the Z-ring. Here, we show the crystal structure of ZapA (11.6 kDa) from Pseudomonas aeruginosa at 2.8 A resolution. The electron density reveals two dimers associating via an extensive C-terminal coiled-coil protrusion to form an elongated anti-parallel tetramer. In solution, ZapA exists in a dimer-tetramer equilibrium that is strongly correlated with concentration. An increase in concentration promotes formation of the higher oligomeric state. The dimer is postulated to be the predominant physiological species although the tetramer could become significant if, as FtsZ is integrated into the Z-ring and is cross-linked, the local concentration of the dimer becomes sufficiently high. We also show that ZapA binds FtsZ with an approximate 1:1 molar stoichiometry and that this interaction provokes dramatic FtsZ polymerisation and inter-filament association as well as yielding filaments, single or bundled, more stable and resistant to collapse. Whilst in vitro dynamics of FtsZ are well characterised, its in vivo arrangement within the ultra-structural architecture of the Z-ring is yet to be determined despite being fundamental to cell division. The ZapA dimer has single 2-fold symmetry whilst the bipolar tetramer displays triple 2-fold symmetry. Given the symmetry of these ZapA oligomers and the polar nature of FtsZ filaments, the structure of ZapA carries novel implications for the inherent architecture of the Z-ring in vivo.


Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Fenômenos Fisiológicos Bacterianos , Cristalografia por Raios X , Dimerização , Elétrons , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Ultracentrifugação
17.
Biochemistry ; 42(47): 13848-55, 2003 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-14636052

RESUMO

RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria. Most endoribonucleases have been shown to act distributively; however, Feng et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746-14751] have recently found that RNase E acts via a scanning mechanism. A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E. coli RNase E forms a homotetramer. Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue. The tetrameric assembly of the N-terminal domain of RNase E is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D(2) dihedral symmetry. Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer. This shape, together with limited proteolysis data, suggests that the S1-RNA binding domains of RNase E lie on the periphery of the tetramer. These observations have implications for the structure and function of the RNase E/RNase G ribonuclease family and for the assembly of the E. coli RNA degradosome, in which RNase E is the central component.


Assuntos
Domínio Catalítico , Endorribonucleases/química , Proteínas de Escherichia coli/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Catálise , Quimotripsina/química , Cristalização , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Nanotecnologia , Polirribonucleotídeo Nucleotidiltransferase/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Helicases/química , Proteínas Recombinantes/química , Espalhamento de Radiação , Espectrometria de Massas por Ionização por Electrospray , Raios X
18.
J Am Chem Soc ; 124(47): 14221-6, 2002 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-12440921

RESUMO

The H-bond ((h3)J(NC')) and peptide bond ((1)J(NC')) scalar couplings establish connectivity of the electronic structure in the H-bond chains of proteins. The correlated changes of (h3)J(NC') and (1)J(NC') couplings extend over several peptide groups in the chains. Consequently, the electronic structure of the H-bond chains can affect (h3)J(NC') in a manner that is independent of the local H-bond geometry. By taking this into account, and by using a more complete set of H-bond geometry parameters, we have predicted (h3)J(NC') couplings in the H-bond chains with deviations commensurate to the standard deviations of the experimentally determined values. We have created a comprehensive database of (h3)J(NC') and (1)J(NC') couplings by measuring the coupling constants in ubiquitin (alphabeta-fold) intestinal fatty acid binding protein (beta-barrel) and carp parvalbumin (alpha-helical).


Assuntos
Proteínas de Neoplasias , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas Supressoras de Tumor , Animais , Apoproteínas/química , Carpas , Proteínas de Transporte/química , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Parvalbuminas/química , Estrutura Secundária de Proteína , Ubiquitina/química
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