RESUMO
Caspase inhibition is a promising approach for treating multiple diseases. Using a reconstituted assay and high-throughput screening, we identified a group of nonpeptide caspase inhibitors. These inhibitors share common chemical scaffolds, suggesting the same mechanism of action. They can inhibit apoptosis in various cell types induced by multiple stimuli; they can also inhibit caspase-1-mediated interleukin generation in macrophages, indicating potential anti-inflammatory application. While these compounds inhibit all the tested caspases, kinetic analysis indicates they do not compete for the catalytic sites of the enzymes. The cocrystal structure of one of these compounds with caspase-7 reveals that it binds to the dimerization interface of the caspase, another common structural element shared by all active caspases. Consistently, biochemical analysis demonstrates that the compound abates caspase-8 dimerization. Based on these kinetic, biochemical, and structural analyses, we suggest that these compounds are allosteric caspase inhibitors that function through binding to the dimerization interface of caspases.
Assuntos
Inibidores de Caspase/química , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Sequência de Aminoácidos , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Cristalografia por Raios X/métodos , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucinas/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacosRESUMO
Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + ß fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.
Assuntos
GMP Cíclico/análogos & derivados , Imunidade Inata , Proteínas de Membrana/química , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Sítios de Ligação , GMP Cíclico/metabolismo , Dimerização , Humanos , Ligação de Hidrogênio , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
TRAF1/2 and cIAP1/2 are members of the TNF receptor-associated factor (TRAF) and the inhibitor of apoptosis (IAP) families, respectively. They are critical for canonical and noncanonical NF-kappaB signaling pathways. Here, we report the crystal structures of the TRAF2: cIAP2 and the TRAF1: TRAF2: cIAP2 complexes. A TRAF2 trimer interacts with one cIAP2 both in the crystal and in solution. Two chains of the TRAF2 trimer directly contact cIAP2, and key residues at the interface are confirmed by mutagenesis. TRAF1 and TRAF2 preferentially form the TRAF1: (TRAF2)(2) heterotrimer, which interacts with cIAP2 more strongly than TRAF2 alone. In contrast, TRAF1 alone interacts very weakly with cIAP2. Surprisingly, TRAF1 and one chain of TRAF2 in the TRAF1: (TRAF2)(2): cIAP2 ternary complex mediate interaction with cIAP2. Because TRAF1 is upregulated by many stimuli, it may modulate the interaction of TRAF2 with cIAP1/2, which explains regulatory roles of TRAF1 in TNF signaling.
Assuntos
Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Conformação Proteica , Fator 1 Associado a Receptor de TNF/química , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/química , Fator 2 Associado a Receptor de TNF/metabolismo , Sequência de Aminoácidos , Animais , Proteína 3 com Repetições IAP de Baculovírus , Linhagem Celular , Cristalografia por Raios X , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese , NF-kappa B/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Fator 1 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/genética , Ubiquitina-Proteína LigasesRESUMO
The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.
Assuntos
Canais de Potássio/química , Canais de Potássio/metabolismo , Vibrio parahaemolyticus/química , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Ativação do Canal Iônico , Transporte de Íons , Modelos Moleculares , Dados de Sequência Molecular , Potássio/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: The autosomal recessive hyper-IgE syndrome (HIES) caused by dedicator of cytokinesis 8 (DOCK8) deficiency shares clinical features with autosomal dominant HIES because of signal transducer and activator of transcription 3 (STAT3) mutations, including recurrent infections and mucocutaneous candidiasis, which are suggestive of TH17 cell dysfunction. The mechanisms underlying this phenotypic overlap are unclear. OBJECTIVE: We sought to elucidate common mechanisms operating in the different forms of HIES. METHODS: We analyzed the differentiation of CD4+ TH cell subsets in control and DOCK8-deficient subjects. We also examined the role of DOCK8 in regulating STAT3 activation in T cells. TH cell differentiation was analyzed by ELISA, flow cytometry, and real-time PCR measurements of cytokines and TH cell transcription factors. The interaction of DOCK8 and STAT3 signaling pathways was examined by using flow cytometry, immunofluorescence, coimmunoprecipitation, and gene expression analysis. RESULTS: There was a profound block in the differentiation of DOCK8-deficient naive CD4+ T cells into TH17 cells. A missense mutation that disrupts DOCK8 guanine nucleotide exchange factor (GEF) activity while sparing protein expression also impaired TH17 cell differentiation. DOCK8 constitutively associated with STAT3 independent of GEF activity, whereas it regulated STAT3 phosphorylation in a GEF activity-dependent manner. DOCK8 also promoted STAT3 translocation to the nucleus and induction of STAT3-dependent gene expression. CONCLUSION: DOCK8 interacts with STAT3 and regulates its activation and the outcome of STAT3-dependent TH17 differentiation. These findings might explain the phenotypic overlap between DOCK8 deficiency and autosomal dominant HIES.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/imunologia , Síndromes de Imunodeficiência/imunologia , Fator de Transcrição STAT3/imunologia , Células Th17/imunologia , Autoanticorpos/imunologia , Diferenciação Celular , Criança , Pré-Escolar , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Lactente , Células Jurkat , Masculino , Mutação , Fosforilação , Transporte Proteico , Fator de Transcrição STAT3/metabolismoRESUMO
The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with beta subunits (Kv betas), and certain Kv betas, for example Kv beta 1, have an N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kv beta 1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channel activity by binding to Kv beta 1. A crystal structure of the Kv beta-cortisone complex was solved to 1.82-A resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kv beta. The new mode of channel modulation may be explored by native or synthetic ligands to fine-tune cellular excitability.
Assuntos
Cortisona/farmacologia , Canal de Potássio Kv1.2/metabolismo , Superfamília Shaker de Canais de Potássio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Cortisona/química , Cristalografia por Raios X , Humanos , Canal de Potássio Kv1.2/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Ratos , Superfamília Shaker de Canais de Potássio/metabolismo , XenopusRESUMO
The NF-κB essential modulator (NEMO) is the scaffolding subunit of the inhibitor of κB kinase (IKK) holocomplex and is required for the activation of the catalytic IKK subunits, IKKα and IKKß, during the canonical inflammatory response. Although structures of shorter constructs of NEMO have been solved, efforts to elucidate the full-length structure of NEMO have proved difficult due to its apparent high conformational plasticity. To better characterize the gross dimensions of full-length NEMO, we employed in-line size exclusion chromatography-small-angle X-ray scattering. We show that NEMO adopts a more compact conformation (Dmax=320Å) than predicted for a fully extended coiled-coil structure (>500Å). In addition, we map a region of NEMO (residues 112-150) in its coiled-coil 1 domain that impedes the binding of linear (M1-linked) di-ubiquitin to its coiled-coil 2-leucine zipper ubiquitin binding domain. This ubiquitin binding inhibition can be overcome by a longer chain of linear, but not K63-linked polyubiquitin. Collectively, these observations suggest that NEMO may be auto-inhibited in the resting state by intramolecular interactions and that during signaling, NEMO may be allosterically activated by binding to long M1-linked polyubiquitin chains.
Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Sítios de Ligação , Humanos , Quinase I-kappa B/química , Ligação Proteica , Conformação Proteica , Transdução de Sinais , UbiquitinaçãoRESUMO
L-Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway of plants and micro-organisms. The aspartate pathway produces fully one-quarter of the naturally occurring amino acids, but is not found in humans or other eukaryotic organisms, making ASADH an attractive target for the development of new antibacterial, fungicidal, or herbicidal compounds. We have determined the structure of ASADH from Vibrio cholerae in two states; the apoenzyme and a complex with NADP, and a covalently bound active site inhibitor, S-methyl-L-cysteine sulfoxide. Upon binding the inhibitor undergoes an enzyme-catalyzed reductive demethylation leading to a covalently bound cysteine that is observed in the complex structure. The enzyme is a functional homodimer, with extensive intersubunit contacts and a symmetrical 4-amino acid bridge linking the active site residues in adjacent subunits that could serve as a communication channel. The active site is essentially preformed, with minimal differences in active site conformation in the apoenzyme relative to the ternary inhibitor complex. The conformational changes that do occur result primarily from NADP binding, and are localized to the repositioning of two surface loops located on the rim at opposite sides of the NADP cleft.
Assuntos
Aspartato-Semialdeído Desidrogenase/química , Aspartato-Semialdeído Desidrogenase/metabolismo , Cisteína/análogos & derivados , Vibrio cholerae/enzimologia , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cisteína/química , Cisteína/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Metilação , Modelos Moleculares , NADP/metabolismo , Conformação ProteicaRESUMO
Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome. The activated caspases process proinflammatory cytokines IL-1ß and IL-18 and induce the inflammatory form of cell death pyroptosis. Here we show that AIM PYD (AIM2(PYD)) self-oligomerizes. We notice significant sequence homology of AIM2(PYD) with the hydrophobic patches of death effector domain (DED)-containing proteins and confirm that mutations on these residues disrupt AIM2(PYD) self-association. The crystal structure at 1.82Å resolution of such a mutant, F27G of AIM2(PYD), shows the canonical six-helix (H1-H6) bundle fold in the death domain superfamily. In contrast to the wild-type AIM2(PYD) structure crystallized in fusion with the large maltose-binding protein tag, the H2-H3 region of the AIM2(PYD) F27G is well defined with low B-factors. Structural analysis shows that the conserved hydrophobic patches engage in a type I interaction that has been observed in DED/DED and other death domain superfamily interactions. While previous mutagenesis studies of PYDs point to the involvement of charged interactions, our results reveal the importance of hydrophobic interactions in the same interfaces. These centrally localized hydrophobic residues within fairly charged patches may form the hot spots in AIM2(PYD) self-association and may represent a common mode of PYD/PYD interactions in general.
Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Ligação a DNA , Humanos , Inflamassomos/metabolismo , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Animais , Linfócitos B/metabolismo , Caspases/metabolismo , Catálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-rel , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.
Assuntos
Proteína de Domínio de Morte Associada a Fas/química , Mutação , Receptor fas/química , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Síndrome Linfoproliferativa Autoimune/genética , Caspase 8/química , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Receptor fas/genéticaRESUMO
The crystal structure of nucleotide-free yeast F(1) ATPase has been determined at a resolution of 3.6 A. The overall structure is very similar to that of the ground state enzyme. In particular, the beta(DP) and beta(TP) subunits both adopt the closed conformation found in the ground state structure despite the absence of bound nucleotides. This implies that interactions between the gamma and beta subunits are as important as nucleotide occupancy in determining the conformational state of the beta subunits. Furthermore, this result suggests that for the mitochondrial enzyme, there is no state of nucleotide occupancy that would result in more than one of the beta subunits adopting the open conformation. The adenine-binding pocket of the beta(TP) subunit is disrupted in the apoenzyme, suggesting that the beta(DP) subunit is responsible for unisite catalytic activity.
Assuntos
Nucleotídeos/metabolismo , Conformação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The crystal structure of yeast mitochondrial F(1) ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the alpha(3)beta(3) sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F(1) ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F(1) ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F(1) ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.
Assuntos
Dobramento de Proteína , ATPases Translocadoras de Prótons/química , Proteínas de Saccharomyces cerevisiae/química , Difosfato de Adenosina/química , Animais , Catálise , Domínio Catalítico , Bovinos , Mitocôndrias/enzimologia , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/enzimologiaRESUMO
The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.
Assuntos
Mitocôndrias/enzimologia , ATPases Translocadoras de Prótons/química , Adenosina Trifosfatases/química , Proliferação de Células , Clorofórmio , Cromatografia de Afinidade , Cromatografia em Gel , Códon , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Histidina/química , Modelos Genéticos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismoRESUMO
A genetically modified (His6-tagged) form of the mitochondrial F1-ATPase (MW = 370 kDa) has been purified from the yeast Saccharomyces cerevisiae and crystallized in the presence of polyethelene glycol (PEG) 6000 as a precipitant, 1 mM NiCl2, 1 mM Mg AMP-PNP and 50 microM Mg ADP. X-ray diffraction data were obtained on three separate occasions using synchrotron radiation, with a progression in the quality of the diffraction data, which improved from 3.3 to 3.0 to 2.8 A. On the second occasion, the diffraction was improved by a crystal-annealing procedure. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 110.6, b = 294.2, c = 190.4 A, beta = 101.6 degrees. The asymmetric unit contains three molecules of yeast F1, with a corresponding volume per protein weight (VM) of 2.8 A3 Da(-1) and a solvent content of 55%.