RESUMEN
The cGAS/STING pathway triggers inflammation upon diverse cellular stresses such as infection, cellular damage, aging, and diseases. STING also triggers noncanonical autophagy, involving LC3 lipidation on STING vesicles through the V-ATPase-ATG16L1 axis, as well as induces cell death. Although the proton pump V-ATPase senses organelle deacidification in other contexts, it is unclear how STING activates V-ATPase for noncanonical autophagy. Here we report a conserved channel function of STING in proton efflux and vesicle deacidification. STING activation induces an electron-sparse pore in its transmembrane domain, which mediates proton flux in vitro and the deacidification of post-Golgi STING vesicles in cells. A chemical ligand of STING, C53, which binds to and blocks its channel, strongly inhibits STING-mediated proton flux in vitro. C53 fully blocks STING trafficking from the ER to the Golgi, but adding C53 after STING arrives at the Golgi allows for selective inhibition of STING-dependent vesicle deacidification, LC3 lipidation, and cell death, without affecting trafficking. The discovery of STING as a channel opens new opportunities for selective targeting of canonical and noncanonical STING functions.
Asunto(s)
Autofagia , Protones , Autofagia/fisiología , Canales Iónicos/genética , Muerte Celular , Adenosina TrifosfatasasRESUMEN
The spike protein (S) of SARS-CoV-2 is the major target of neutralizing antibodies and vaccines. Antibodies that target the receptor-binding domain (RBD) of S have high potency in preventing viral infection. The ongoing evolution of SARS-CoV-2, especially mutations occurring in the RBD of new variants, has severely challenged the development of neutralizing antibodies and vaccines. Here, a murine monoclonal antibody (mAb) designated E77 is reported which engages the prototype RBD with high affinity and potently neutralizes SARS-CoV-2 pseudoviruses. However, the capability of E77 to bind RBDs vanishes upon encountering variants of concern (VOCs) which carry the N501Y mutation, such as Alpha, Beta, Gamma and Omicron, in contrast to its performance with the Delta variant. To explain the discrepancy, cryo-electron microscopy was used to analyze the structure of an RBD-E77 Fab complex, which reveals that the binding site of E77 on RBD belongs to the RBD-1 epitope, which largely overlaps with the binding site of human angiotensin-converting enzyme 2 (hACE2). Both the heavy chain and the light chain of E77 interact extensively with RBD and contribute to the strong binding of RBD. E77 employs CDRL1 to engage Asn501 of RBD and the Asn-to-Tyr mutation could generate steric hindrance, abolishing the binding. In sum, the data provide the landscape for an in-depth understanding of immune escape of VOCs and rational antibody engineering against emerging variants of SARS-CoV-2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Animales , Ratones , Microscopía por Crioelectrón , Anticuerpos NeutralizantesRESUMEN
The FAM46 (also known as TENT5) proteins are noncanonical poly(A) polymerases (PAPs) implicated in regulating RNA stability. The regulatory mechanisms of FAM46 are poorly understood. Here, we report that the nuclear protein BCCIPα, but not the alternatively spliced isoform BCCIPß, binds FAM46 and inhibits their PAP activity. Unexpectedly, our structures of the FAM46A/BCCIPα and FAM46C/BCCIPα complexes show that, despite sharing most of the sequence and differing only at the C-terminal portion, BCCIPα adopts a unique structure completely different from BCCIPß. The distinct C-terminal segment of BCCIPα supports the adoption of the unique fold but does not directly interact with FAM46. The ß sheets in BCCIPα and FAM46 pack side by side to form an extended ß sheet. A helix-loop-helix segment in BCCIPα inserts into the active site cleft of FAM46, thereby inhibiting the PAP activity. Our results together show that the unique fold of BCCIPα underlies its interaction with and functional regulation of FAM46.
Asunto(s)
Proteínas Nucleares , Dominio Catalítico , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Polinucleotido Adenililtransferasa/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMEN
2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Transducción de Señal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , ADN , Inmunidad InnataRESUMEN
The cGAS-cGAMP-STING pathway is an important innate immune signaling cascade responsible for the sensing of abnormal cytosolic double-stranded DNA (dsDNA), which is a hallmark of infection or cancers. Recently, tremendous progress has been made in the understanding of the STING activation mechanism from various aspects. In this review, the molecular mechanism of activation of STING protein based on its structural features is briefly discussed. The underlying molecular mechanism of STING activation will enable us to develop novel therapeutics to treat STING-associated diseases and understand how STING has evolved to eliminate infection and maintain immune homeostasis in innate immunity.
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Inmunidad Innata , Proteínas de la Membrana , Citosol/metabolismo , ADN , Proteínas de la Membrana/genética , Transducción de SeñalRESUMEN
Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria1-5. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria1,6,7. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways8,9. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53)10, promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STING than either ligand alone.
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Proteínas de la Membrana , Nucleótidos Cíclicos , Proteínas de Ciclo Celular , Microscopía por Crioelectrón , Fosfatos de Dinucleósidos/metabolismo , Humanos , Inmunidad Innata , Ligandos , Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/metabolismo , Proteínas Supresoras de TumorRESUMEN
Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.
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Proteínas del Tejido Nervioso/ultraestructura , Neuropilina-1/ultraestructura , Receptores de Superficie Celular/ultraestructura , Semaforina-3A/ultraestructura , Animales , Células COS , Chlorocebus aethiops , Microscopía por Crioelectrón , Células HEK293 , Humanos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/genética , Neuropilina-1/aislamiento & purificación , Neuropilina-1/metabolismo , Unión Proteica/genética , Dominios Proteicos/genética , Multimerización de Proteína/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Semaforina-3A/genética , Semaforina-3A/aislamiento & purificación , Semaforina-3A/metabolismoRESUMEN
FAM46C, a non-canonical poly(A) polymerase, is frequently mutated in multiple myeloma. Loss of function of FAM46C promotes cell survival of multiple myeloma, suggesting a tumor-suppressive role. FAM46C is also essential for fastening sperm head and flagellum, indispensable for male fertility. The molecular mechanisms of these functions of FAM46C remain elusive. We report the crystal structure of FAM46C to provide the basis for its poly(A) polymerase activity and rationalize mutations associated with multiple myeloma. In addition, we found that FAM46C interacts directly with the serine/threonine kinase Plk4, the master regulator of centrosome duplication. We present the structure of FAM46C in complex with the Cryptic Polo-Box 1-2 domains of Plk4. Our structure-based mutational analyses show that the interaction with Plk4 recruits FAM46C to centrosomes. Our data suggest that Plk4-mediated localization of FAM46C enables its regulation of centrosome structure and functions, which may underlie the roles for FAM46C in cell proliferation and sperm development.
Asunto(s)
Nucleotidiltransferasas/química , Proteínas Serina-Treonina Quinasas/química , Sitios de Unión , Línea Celular Tumoral , Centrosoma/química , Centrosoma/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Mutación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The cyclic dinucleotide (CDN)-stimulator of interferon genes (STING) pathway plays an important role in the detection of viral and bacterial pathogens in animals. Previous studies have shown that the metazoan second messenger cyclic [G(2',5')pA(3',5')p] (2',3'-cGAMP) generated by cyclic GMP-AMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3',3'-cGAMP. Despite recent progress indicating that the CDN-binding domain (CBD) of dimeric STING binds asymmetric 2',3'-cGAMP preferentially over symmetric 3',3'-CDNs, it remains an open question whether STING molecules, such as human STING, adopt a symmetric dimeric conformation to efficiently engage its asymmetric ligand. Here, structural studies of the CBD from porcine STING (STINGCBD) in complex with CDNs at 1.76-2.6 Å resolution revealed that porcine STINGCBD, unlike its human and mouse counterparts, can adopt an asymmetric ligand-binding pocket to accommodate the CDNs. We observed that the extensive interactions and shape complementarity between asymmetric 2',3'-cGAMP and the ligand-binding pocket make it the most preferred ligand for porcine STING and that geometry constraints limit the binding between symmetric 3',3'-CDN and porcine STING. The ligand-discrimination mechanism of porcine STING observed here expands our understanding of how the CDN-STING pathway is activated and of its role in antiviral defense.
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Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Estructura Molecular , Unión Proteica , PorcinosRESUMEN
The GIPC family adaptor proteins mediate endocytosis by tethering cargo proteins to the myosin VI motor. The structural mechanisms for the GIPC/cargo and GIPC/myosin VI interactions remained unclear. PlexinD1, a transmembrane receptor that regulates neuronal and cardiovascular development, is a cargo of GIPCs. GIPC-mediated endocytic trafficking regulates PlexinD1 signaling. Here, we unravel the mechanisms of the interactions among PlexinD1, GIPCs and myosin VI by a series of crystal structures of these proteins in apo or bound states. GIPC1 forms a domain-swapped dimer in an autoinhibited conformation that hinders binding of both PlexinD1 and myosin VI. PlexinD1 binding to GIPC1 releases the autoinhibition, promoting its interaction with myosin VI. GIPCs and myosin VI interact through two distinct interfaces and form an open-ended alternating array. Our data support that this alternating array underlies the oligomerization of the GIPC/Myosin VI complexes in solution and cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Multimerización de Proteína , Animales , Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The type VI secretion system (T6SS) has recently been demonstrated to mediate interbacterial competition and to discriminate between self and nonself. T6SS(+) bacteria employ toxic effectors to inhibit rival cells and concurrently use effector cognate immunity proteins to protect their sibling cells. The effector and immunity pairs (E-I pairs) endow the bacteria with a great advantage in niche competition. Tle4-Tli4 (PA1510-PA1509) is a newly identified E-I pair that is controlled by H2-T6SS in Pseudomonas aeruginosa. Tle4 exhibits phospholipase activity, which destroys the cell membrane of rival cells, and the periplasm-located Tli4 in donor cells eliminates this toxic effect of Tle4. In this paper, the structure of the Tle4-Tli4 complex is reported at 1.75â Å resolution. Tle4 consists of two domains: a conserved α/ß-hydrolase domain and an unusual cap domain in which two lid regions (lid1 and lid2) display a closed conformation that buries the catalytic triad in a deep funnel. Tli4 also displays a two-domain structure, in which a large lobe and a small lobe form a crab claw-like conformation. Tli4 uses this crab claw to grasp the cap domain of Tle4, especially the lid2 region, which prevents the interfacial activation of Tle4 and thus causes enzymatic dysfunction of Tle4 in sister cells.
Asunto(s)
Proteínas Bacterianas/química , Sistemas de Secreción Bacterianos , Pseudomonas aeruginosa/química , Cristalografía por Rayos X , Hidrolasas/química , Modelos Moleculares , Fosfolipasas/química , Conformación Proteica , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/fisiologíaRESUMEN
Cyclic dinucleotides are a newly expanded class of second messengers that contribute to the regulation of multiple different pathways in bacterial, eukaryotic, and archaeal cells. The recently identified Vibrio cholerae dinucleotide cyclase (DncV, the gene product of VC0179) can generate three different cyclic dinucleotides and preferentially synthesize a hybrid cyclic-GMP-AMP. Here, we report the crystal structural and functional studies of DncV. We unexpectedly observed a 5-methyltetrahydrofolate diglutamate (5MTHFGLU2) molecule bound in a surface pocket opposite the nucleotide substrate-binding groove of DncV. Subsequent mutagenesis and functional studies showed that the enzymatic activity of DncV is regulated by folate-like molecules, suggesting the existence of a signaling pathway that links folate-like metabolism cofactors to the regulation of cyclic dinucleotide second messenger synthesis. Sequence analysis showed that the residues involved in 5MTHFGLU2 binding are highly conserved in DncV orthologs, implying the presence of this regulation mechanism in a wide variety of bacteria.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ácido Fólico/análogos & derivados , Vibrio cholerae/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Ácido Fólico/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Vibrio cholerae/químicaRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Calcio/metabolismo , Unión ProteicaRESUMEN
Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to inject effector proteins into rival cells in niche competition. Tse3, one of the effectors of T6SS, is delivered into the periplasm of recipient cells. Tse3 functions as a muramidase that degrades the ß-1,4-linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan, thus leading to lysis of the recipient cells and providing a competitive advantage to the donor cells. Here, the preliminary crystallographic study of Tse3 is reported. A crystal of Tse3 diffracted to 1.5â Å resolution. It belonged to space group C121, with unit-cell parameters a = 166.99, b = 70.13, c = 41.94â Å, α = 90.00, ß = 90.52, γ = 90.00° and one molecule per asymmetric unit.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia MolecularRESUMEN
Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Cullin/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/metabolismo , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/genética , Complejo del Señalosoma COP9 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas Cullin/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Genes Ligados a X/genética , Células HEK293 , Humanos , Immunoblotting , Discapacidad Intelectual/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Microscopía Fluorescente , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Interferencia de ARN , UbiquitinaciónRESUMEN
Tse1 (Tse is type VI secretion exported), an effector protein produced by Pseudomonas aeruginosa, is an amidase that hydrolyses the γ-D-glutamyl-DAP (γ-D-glutamyl-L-meso-diaminopimelic acid) linkage of the peptide bridge of peptidoglycan. P. aeruginosa injects Tse1 into the periplasm of recipient cells, degrading their peptidoglycan, thereby helping itself to compete with other bacteria. Meanwhile, to protect itself from injury by Tse1, P. aeruginosa expresses the cognate immunity protein Tsi1 (Tsi is type VI secretion immunity) in its own periplasm to inactivate Tse1. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6-148)-Tsi1-(20-end) complex at 1.4 Å and 1.6 Å (1 Å=0.1 nm) resolutions respectively. The Tse1 structure adopts a classical papain-like α+ß fold. A cysteine-histidine catalytic diad is identified in the reaction centre of Tse1 by structural comparison and mutagenesis studies. Tsi1 binds Tse1 tightly. The HI loop (middle finger tip) from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1, and CD, EF, JK and LM loops (thumb, index finger, ring finger and little finger tips) interact with Tse1, thus blocking the binding of enzyme to peptidoglycan. The catalytic and inhibition mechanisms provide new insights into how P. aeruginosa competes with others and protects itself.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Mutagénesis Sitio-Dirigida , N-Acetil Muramoil-L-Alanina Amidasa/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
STING functions as both an adaptor protein signaling cytoplasmic double-stranded DNA and a direct immunosensor of cyclic diguanylate monophosphate (c-di-GMP). The crystal structures of the C-terminal domain of human STING (STING(CTD)) and its complex with c-di-GMP reveal how STING recognizes c-di-GMP. In response to c-di-GMP binding, two surface loops, which serve as a gate and latch of the cleft formed by the dimeric STING(CTD), undergo rearrangements to interact with the ligand.
Asunto(s)
Proteínas de la Membrana/química , GMP Cíclico/análogos & derivados , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Interacciones Huésped-Patógeno , Legionella pneumophila/patogenicidad , Proteínas de Unión al GTP rab1/metabolismo , Secuencia de Aminoácidos , Cristalización , Inhibidores de Disociación de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Legionella pneumophila/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Análisis de Secuencia de Proteína , Proteínas de Unión al GTP rab1/química , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Cullin 4B (CUL4B) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes. Recent studies have revealed that germ-line mutations in CUL4B can cause mental retardation, short stature, and many other abnormalities in humans. Identifying specific CUL4B substrates will help to better understand the physiological functions of CUL4B. Here, we report the identification of peroxiredoxin III (PrxIII) as a novel substrate of the CUL4B ubiquitin ligase complex. Two-dimensional gel electrophoresis coupled with mass spectrometry showed that PrxIII was among the proteins up-regulated in cells after RNAi-mediated CUL4B depletion. The impaired degradation of PrxIII observed in CUL4B knockdown cells was confirmed by Western blot. We further demonstrated that DDB1 and ROC1 in the DDB1-CUL4B-ROC1 complex are also indispensable for the proteolysis of PrxIII. In addition, the degradation of PrxIII is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of PrxIII. Furthermore, we observed a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells, which was associated with increased resistance to hypoxia and H(2)O(2)-induced apoptosis. These findings are discussed with regard to the known function of PrxIII as a ROS scavenger and the high endogenous ROS levels required for neural stem cell proliferation. Together, our study has identified a specific target substrate of CUL4B ubiquitin ligase that may have significant implications for the pathogenesis observed in patients with mutations in CUL4B.
Asunto(s)
Proteínas Cullin/metabolismo , Discapacidad Intelectual/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Peroxiredoxina III/metabolismo , Ubiquitinación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Proliferación Celular/efectos de los fármacos , Proteínas Cullin/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Mutación de Línea Germinal , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/patología , Oxidantes/farmacología , Peroxiredoxina III/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin alpha1, alpha3, and alpha5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore, the nuclear localization of CUL4B mediated by NLS is critical for its normal function in cell proliferation.