RESUMEN
The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.
Asunto(s)
Proteína 10 de la LLC-Linfoma de Células B , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Dominios Proteicos , Multimerización de Proteína , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteína 10 de la LLC-Linfoma de Células B/química , Proteína 10 de la LLC-Linfoma de Células B/genética , Humanos , Cristalografía por Rayos X , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Caspasas/metabolismo , Caspasas/químicaRESUMEN
NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail, while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly employs the C-terminal arm including the K63 linkage residue. These studies uncover the energetics and geometry for mutual recognition of NEMO and diubiquitins.
Asunto(s)
Quinasa I-kappa B/química , Ubiquitinas/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Cristalografía por Rayos X , Predisposición Genética a la Enfermedad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , FN-kappa B/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Relación Estructura-Actividad , Ubiquitinas/metabolismoRESUMEN
MyD88, IRAK4 and IRAK2 are critical signalling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88-IRAK4-IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signalling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88-IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex, which are confirmed by mutagenesis and previously identified signalling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88-IRAK4-IRAK2 complex provides a template for Toll signalling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1 , Modelos Moleculares , Factor 88 de Diferenciación Mieloide , Receptores de Interleucina-1/fisiología , Transducción de Señal , Receptores Toll-Like/fisiología , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/química , Factor 88 de Diferenciación Mieloide/metabolismo , Estructura Terciaria de Proteína , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Apoptosis is an important process to maintain cellular homeostasis. Deregulated apoptosis has linked to a number of diseases, such as inflammatory diseases, neurodegenerative disorder, and cancers. A major signaling complex in the death receptor signaling pathway leading to apoptosis is death-induced signaling complex (DISC), which is regulated mainly by death effector domain (DED)-containing proteins. There are seven DED-containing proteins in human, including FADD, c-FLIP, caspase-8, caspase-10, DEDD, DEDD2, and PEA-15. The main players in DISC formation employ tandem DEDs for regulating signaling complex formation. The regulatory mechanism of signaling complex formation is important and yet remains unclear. Interestingly, three caspase recruitment domain (CARD)-containing members, which belong to the same DD superfamily as DED-containing proteins, also contains similar tandem CARDs. Recent structural studies have shown that tandem CARDs are essential for the formation of a helical signaling complex. This review summarizes recent structural studies on DED-containing proteins and especially discusses the studies on tandem DEDs and tandem CARDs, which suggest new mechanisms of signaling complex assembly.
Asunto(s)
Apoptosis , Proteínas Adaptadoras de Señalización CARD/fisiología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/fisiología , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/fisiología , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transducción de Señal , Homología Estructural de ProteínaRESUMEN
CARD subfamily is the second largest subfamily in the DD superfamily that plays important roles in regulating various signaling pathways, including but not limited to NF-kB activation signaling, apoptosis signaling and inflammatory signaling. The CARD subfamily contains 33 human CARD-containing proteins, regulating the assembly of many signaling complexes, including apoptosome, inflammsome, nodosome, the CBM complex, PIDDosome, the TRAF2 complex, and the MAVS signalosome, by homotypic CARD-CARD interactions. The mechanism of how CARDs find the right binding partner to form a specific complex remains unclear. This review uses different classification schemes to update the classification of CARD-containing proteins. Combining the classification based on domain structures, functions, associated signaling complexes, and roles would help better understand the structural and function diversity of CARD-containing proteins. This review also summarizes recent structural studies on CARDs. Especially, the CARD-containing complexes can be divided into the homodimeric, heterodimeric, oligomeric, filamentous CARD complexes and the CARD-ubiquitin complex. This review will give an overview of the versatile roles of CARDs in regulating signaling transduction, as well as the therapeutic drugs targeting CARD-containing proteins.
Asunto(s)
Apoptosis , Proteínas Adaptadoras de Señalización CARD/fisiología , FN-kappa B/metabolismo , Humanos , Inflamación/metabolismo , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Receptores de Muerte Celular/fisiología , Transducción de SeñalRESUMEN
Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.
Asunto(s)
Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Caspasa 8 , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Células HEK293 , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de SeñalRESUMEN
Polyubiquitination is important in controlling NF-κB signaling. Excessive NF-κB activity has been linked to inflammatory disorders and autoimmune diseases, while ABIN1 could attenuate NF-κB activation to maintain immune homeostasis by utilizing UBAN to recognize linear (M1)-linked polyubiquitinated NF-κB activation mediators, including NEMO, IRAK1 and RIP1. PolyUb-mediated UBAN recruitment remains undetermined, since the recognition studies focused mostly on di-ubiquitin (diUb). Here we report three crystal structures of human ABIN1 UBAN (hABIN1UBAN) in complex with M1-linked diUb, triUb, and tetraUb, respectively. Notably, the hABIN1UBAN:diUb structure reveals that a diUb randomly binds one of the Ub-binding sites of the hABIN1UBAN dimer and leaves the other site vacant. Together with the ITC and gel-filtration analyses, we found that M1-triUb and M1-tetraUb adopt two unique conformations, instead of an elongated one, and they preferentially use the N-terminal two-Ub unit to bind the primary Ub-binding site of a hABIN1UBAN dimer and the C-terminal two-Ub unit to bind the secondary Ub-binding site of another hABIN1UBAN dimer. Especially, our results suggest that two ABIN1UBAN dimers cooperatively bind two UBAN-binding units of a tetraUb or vice versa. Since the UBAN family members share a conserved diUb-binding mode, our results suggest that M1-polyUb modification allows multiple copies of the two-tandem Ub unit to simultaneously coordinate multiple and/or different binding partners to increase their local concentrations and to facilitate the formation of a large signaling complex. Our study provides a structural-functional glimpse of M1-polyUb as a multiple-molecule binding platform to exert its intrinsic structural plasticity in mediating cellular signaling.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Ubiquitinación , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Homología de Secuencia , Ubiquitinas/químicaRESUMEN
X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.
Asunto(s)
Conformación Proteica , Proteína Inhibidora de la Apoptosis Ligada a X/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Tumor necrosis factor (TNF) receptor (TNFR) associated factor 6 (TRAF6) is a unique member of the TRAF family of adaptor proteins that is involved in both the TNF receptor superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily signal transduction pathways. The ability to mediate signals from both families of receptors implicates TRAF6 as an important regulator of a diverse range of physiological processes such as innate and adaptive immunity, bone metabolism, and the development of lymph nodes, mammary glands, skin, and the central nervous system. This chapter will highlight the structural and biochemical studies of TRAF6 in receptor interactions and discuss the potential for peptidomimetic drug application based on TRAF6 receptor binding motif.
Asunto(s)
Factor 6 Asociado a Receptor de TNF/química , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Humanos , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/fisiologíaRESUMEN
Recognition of linear polyubiquitin by specific ubiquitin-binding proteins plays an important role in mediating nuclear factor-κB (NF-κB) signaling. A20 binding proteins, ABINs, recognize linear polyubiquitin and A20 through UBAN and AHD1, respectively, for the inhibition of NF-κB activation. Here we report the crystal structure of the AHD1-UBAN fragment of ABIN2 in complex with linear tri-ubiquitin, which reveals a 2:1 stoichiometry of the complex. Structural analyses together with mutagenesis, pull-down, and isothermal titration calorimetry assays show that the hABIN2:tri-ubiquitin interaction is mainly through the primary ubiquitin-binding site, and also through the secondary ubiquitin-binding site under a high local protein concentration. Surprisingly, three ubiquitin units could form a right-handed helical trimer to bridge two ABIN2 dimers. The residues around the M1-linkage are crucial for ABIN2 to recognize tri-ubiquitin. The tri-ubiquitin bridging two ABIN2 dimers model suggests a possible higher-order signaling complex assembled between M1-linked polyubiquitinated proteins, ubiquitin-binding proteins, and effector signaling proteins in signal transduction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Poliubiquitina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Death domain (DD)-fold assemblies play a crucial role in regulating the signaling to cell survival or death. Here we report the crystal structure of the caspase recruitment domain (CARD)-CARD disk of the human apoptosome. The structure surprisingly reveals that three 1:1 Apaf-1:procaspase-9 CARD protomers form a novel helical DD-fold assembly on the heptameric wheel-like platform of the apoptosome. The small-angle X-ray scattering and multi-angle light scattering data also support that three protomers could form an oligomeric complex similar to the crystal structure. Interestingly, the quasi-equivalent environment of CARDs could generate different quaternary CARD assemblies. We also found that the type II interaction is conserved in all DD-fold complexes, whereas the type I interaction is found only in the helical DD-fold assemblies. This study provides crucial insights into the caspase activation mechanism, which is tightly controlled by a sophisticated and highly evolved CARD assembly on the apoptosome, and also enables better understanding of the intricate DD-fold assembly.
Asunto(s)
Apoptosomas/química , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caspasa 9/metabolismo , Apoptosis , Apoptosomas/metabolismo , Factor Apoptótico 1 Activador de Proteasas/química , Caspasa 9/química , Cristalografía por Rayos X , Activación Enzimática , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo PequeñoRESUMEN
Volvatoxin A2, a pore-forming cardiotoxic protein, was isolated from the edible mushroom Volvariella volvacea. Previous studies have demonstrated that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin A1 at a volvatoxin A2/volvatoxin A1 molar ratio of 2. In this study, we investigated the molecular mechanism by which volvatoxin A1 inhibits the cytotoxicity of volvatoxin A2. Volvatoxin A1 by itself was found to be nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activities of volvatoxin A2 at molar ratios of 2 or lower. Interestingly, volvatoxin A1 contains 393 amino acid residues that closely resemble a tandem repeat of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic alpha-helices but it lacks a heparin-binding site. This suggests that volvatoxin A1 may interact with volvatoxin A2 but not with the cell membrane. By using confocal microscopy, it was demonstrated that volvatoxin A1 could not bind to the cell membrane; however, volvatoxin A1 could inhibit binding of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide competition assay and in conjunction with pull-down and co-pull-down experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may form a complex. Our results suggest that this occurs via the interaction of one molecule of volvatoxin A1, which contains two amphipathic alpha-helices, with two molecules of volvatoxin A2, each of which contains one amphipathic alpha-helix. Taken together, the results of this study reveal a novel mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin A2 via direct interaction, and potentially provide an exciting new strategy for chemotherapy.
Asunto(s)
Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Micotoxinas/antagonistas & inhibidores , Micotoxinas/química , Proteínas/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Secuencia Conservada , Cristalografía por Rayos X , ADN Complementario/genética , Dimerización , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutatión Transferasa/metabolismo , Células HeLa , Hemólisis/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Liposomas/metabolismo , Datos de Secuencia Molecular , Micotoxinas/genética , Micotoxinas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Membrane adhesion and insertion of protein are essential to all organisms, but the underlying mechanisms remain largely unknown. Membrane pore-forming toxins (PFTs) are potential model systems for studying these mechanisms. We have determined the crystal structures of volvatoxin A2 (VVA2), a fungal PFT from Volvariella volvacea, using Br-multiple-wavelength anomalous diffraction (MAD). The VVA2 structures obtained at pH 4.6, pH 5.5 and pH 6.5 were refined to resolutions of 1.42 A, 2.6 A and 3.2 A, respectively. The structures reveal that the VVA2 monomer contains a single alpha/beta domain. Most of the VVA2 surface is occupied by its oligomerization motif and two putative heparin-binding motifs. Residues Ala91 to Ala101 display several conformations at different pH values, which might be under the control of His87. We also found that the shape of one putative heparin-binding motif in VVA2 appears similar to those found in fibroblast growth factors, and the other one displays a linear polypeptide. Our results suggest several possible intermediates of protein assembly in solution and protein adhering to cell membranes before conformational changes. The electron micrographs of VVA2 molecules in solution, at a protein concentration of 1 microg ml(-1), show that they can assemble into filament-like or braid-like oligomers in a pH-dependent way. In addition, the arc-shaped VVA2 structure obtained at pH 6.5 suggests that VVA2 could form a two-layered helical oligomer with 18 subunits per turn. The structures presented here could be used to elucidate the pore-formation mechanisms of VVA2 and its structural neighbors, Cyt toxins from Bacillus thuringiensis.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/ultraestructura , Micotoxinas/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Aniones , Sitios de Unión , Cristalografía por Rayos X , Proteínas Fúngicas/metabolismo , Heparina/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Micotoxinas/metabolismo , Alineación de SecuenciaRESUMEN
Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing activities of thioesterase, esterase, arylesterase, protease, and lysophospholipase. In particular, TAP has stereoselectivity for amino acid derivative substrates, hence it is useful for the kinetic resolution of racemic mixtures of industrial chemicals. In the present work, the crystal structure of native TAP was determined at 1.9A, revealing a minimal SGNH-hydrolase fold. The structure of TAP in complex with a diethyl phosphono moiety (DEP) identified its catalytic triad, Ser10-Asp154-His157, and oxyanion hole, Ser10-Gly44-Asn73. The oxyanion hole of TAP consists of three residues each separated from the other by more than 3.5A, implying that all of them are highly polarized when substrate bound. The catalytic (His)C(epsilon1)-H...O=C hydrogen bond usually plays a role in the catalytic mechanisms of most serine hydrolases, however, there were none present in SGNH-hydrolases. We propose that the existence of the highly polarized tri-residue-constituted oxyanion hole compensates for the lack of a (His)C(epsilon1)-H...O=C hydrogen bond. This suggests that members of the SGNH-hydrolase family may employ a unique catalytic mechanism. In addition, most SGNH-hydrolases have low sequence identities and presently there is no clear criterion to define consensus sequence blocks. Through comparison of TAP and the three SGNH-hydrolase structures currently known, we have identified a unique hydrogen bond network which stabilizes the catalytic center: a newly discovered structural feature of SGNH-hydrolases. We have defined these consensus sequence blocks providing a basis for the sub-classification of SGNH-hydrolases.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lisofosfolipasa/química , Lisofosfolipasa/metabolismo , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Secuencia de Consenso , Secuencia Conservada , Cristalografía por Rayos X , Enlace de Hidrógeno , Hidrolasas/química , Hidrolasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Palmitoil-CoA Hidrolasa/química , Palmitoil-CoA Hidrolasa/metabolismo , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Polyubiquitin chains are regulatory signals for a wide array of biological processes. Recent structural studies reveal novel modes of polyubiquitin chain recognition and implicate the diverse repertoire of interactions in providing the specificity of polyubiquitin recognition.
RESUMEN
Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.
Asunto(s)
Factor 6 Asociado a Receptor de TNF/química , Enzimas Ubiquitina-Conjugadoras/química , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Conformación Proteica , Multimerización de Proteína , Dominios RING Finger , Ubiquitinación , Dedos de ZincRESUMEN
Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Ubiquitinación , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Catálisis , Línea Celular , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Unión al ADN , Escherichia coli/genética , Eliminación de Gen , Glutatión Transferasa/metabolismo , Humanos , Enlace de Hidrógeno , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Riñón/citología , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Poliubiquitina/química , Poliubiquitina/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática , Especificidad por Sustrato , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. It has been thought that small-molecule mimetics of Smac, an endogenous IAP antagonist, might potentiate apoptosis in cancer cells by promoting caspase activation. However, three recent papers, two in Cell (Vince et al., 2007; Varfolomeev et al., 2007) and one in Cancer Cell (Petersen et al., 2007), now report that Smac mimetics primarily kill cancer cells via a different mechanism, the induction of autoubiquitination and degradation of cIAPs, which culminates in TNFalpha-mediated cell death.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/metabolismo , Imitación Molecular , Neoplasias/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Mitocondriales/genética , FN-kappa B/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily, and the pyrin domain (PYD) subfamily is one of the largest domain superfamilies. By mediating homotypic interactions within each domain subfamily, these proteins play important roles in the assembly and activation of apoptotic and inflammatory complexes. In this chapter, we review the molecular complexes assembled by these proteins, the structural and biochemical features of these domains, and the molecular interactions mediated by them. By analyzing the potential molecular basis for the function of these domains, we hope to provide a comprehensive understanding of the function, structure, interaction, and evolution of this important family of domains.
Asunto(s)
Apoptosis/inmunología , Proteínas Adaptadoras de Señalización CARD/inmunología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/inmunología , Evolución Molecular , Complejos Multiproteicos/inmunología , Transducción de Señal/inmunología , Animales , Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Complejos Multiproteicos/genética , Estructura Cuaternaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Transducción de Señal/genética , Relación Estructura-ActividadRESUMEN
Transforming growth factor beta-activated kinase 1 (TAK1), a member of the MAPKKK family, was initially described to play an essential role in the transforming growth factor beta-signaling pathway, but recent evidence has emerged implicating TAK1 in the interleukin (IL)-1 and tumor necrosis factor (TNF) pathways. Notably, two homologous proteins, TAB2 and TAB3, have been identified as adaptors linking TAK1 to the upstream adaptors TRAFs. However, it remains unclear whether the interaction between TAB2/TAB3 and TAK1 is necessary for its kinase activation and subsequent activation of the IKK and MAPK pathways. Here, we characterized the TAB2/TAB3-binding domain in TAK1 and further examined the requirement of this interaction for IL-1, TNF, and RANKL signaling. Through deletion mapping experiments, we demonstrated that the binding motif for TAB2/TAB3 is a non-contiguous region located within the last C-terminal 100 residues of TAK1. However, residues 479-553 of TAK1 appear to be necessary and sufficient for TAB2/TAB3 interaction. Conversely, residues 574-693 of TAB2 were shown to interact with TAK1. A green fluorescent protein fusion protein containing the last 100 residues of TAK1 (TAK1-C100) abolished the interaction of endogenous TAB2/TAB3 with TAK1, the phosphorylation of TAK1, and prevented the activation of IKK and MAPK induced by IL-1, TNF, and RANKL. Furthermore, TAK1-C100 blocked RANKL-induced nuclear accumulation of NFATc1 and consequently osteoclast differentiation consistent with the ability of a catalytically inactive TAK1 to block RANKL-mediated signaling. Significantly, our study provides evidence that the TAB2/TAB3 interaction with TAK1 is crucial for the activation of signaling cascades mediated by IL-1, TNF, and RANKL.