RESUMO
Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (ß-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.
Assuntos
Proteínas de Drosophila , Domínios PDZ , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Desgrenhadas/metabolismo , Proteínas de Drosophila/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Fosfoproteínas/metabolismo , Transdução de SinaisRESUMO
Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.
Assuntos
Proteínas Desgrenhadas/química , Receptores Frizzled/química , Proteínas Wnt/química , beta Catenina/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Clonagem Molecular , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (ß-catenin-dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.
Assuntos
Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Sequência Conservada , Proteínas Desgrenhadas/genética , Humanos , Modelos Moleculares , Mutação/genética , Fosforilação , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Serina/metabolismo , Relação Estrutura-Atividade , Termodinâmica , Via de Sinalização WntRESUMO
PHD fingers are modular domains in chromatin-associated proteins that decode the methylation status of histone H3 tails. A PHD finger signature is found in plant vernalization (VEL) proteins, which function as accessory factors of the Polycomb system to control flowering in Arabidopsis through an epigenetic silencing mechanism. It has been proposed that VEL PHD fingers bind to methylated histone H3 tails to facilitate association of the Polycomb silencing machinery with target genes. Here, we use structural analysis by X-ray crystallography to show that the VEL PHD finger forms the central module of a larger compact tripartite superdomain that also contains a zinc finger and a four-helix bundle. This PHD superdomain fold is only found in one other family, the OBERON proteins, which have multiple functions in Arabidopsis meristems to control plant growth. The putative histone-binding surface of OBERON proteins exhibits the characteristic three-pronged pocket of histone-binding PHD fingers and binds to methylated histone H3 tails. However, that of VEL PHD fingers lacks this architecture and exhibits unusually high positive surface charge. This VEL PHD superdomain neither binds to unmodified nor variously modified histone H3 tails, as demonstrated by isothermal calorimetry and NMR spectroscopy. Instead, the VEL PHD superdomain interacts with negatively charged polymers. Our evidence argues for evolution of a divergent function for the PHD superdomain in vernalization that does not involve histone tail decoding.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Flores , Histonas , Arabidopsis/genética , Arabidopsis/fisiologia , Histonas/metabolismo , Ligação Proteica , Periodicidade , Flores/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologiaRESUMO
FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, FtsK translocates double-stranded DNA until both dif recombination sites are placed at mid cell for subsequent dimer resolution. Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of the motor domain of FtsK while translocating on its DNA substrate. Each subunit of the homo-hexameric ring adopts a unique conformation and one of three nucleotide states. Two DNA-binding loops within four subunits form a pair of spiral staircases within the ring, interacting with the two DNA strands. This suggests that simultaneous conformational changes in all ATPase domains at each catalytic step generate movement through a mechanism related to filament treadmilling. While the ring is only rotating around the DNA slowly, it is instead the conformational states that rotate around the ring as the DNA substrate is pushed through.
Assuntos
DNA Bacteriano/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Translocação Genética/fisiologia , Divisão Celular/fisiologia , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Microscopia Crioeletrônica , DNA/química , DNA Bacteriano/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Conformação ProteicaRESUMO
The Chip/LIM-domain binding protein (LDB)-single-stranded DNA-binding protein (SSDP) (ChiLS) complex controls numerous cell-fate decisions in animal cells, by mediating transcription of developmental control genes via remote enhancers. ChiLS is recruited to these enhancers by lineage-specific LIM-domain proteins that bind to its Chip/LDB subunit. ChiLS recently emerged as the core module of the Wnt enhanceosome, a multiprotein complex that primes developmental control genes for timely Wnt responses. ChiLS binds to NPFxD motifs within Pygopus (Pygo) and the Osa/ARID1A subunit of the BAF chromatin remodeling complex, which could synergize with LIM proteins in tethering ChiLS to enhancers. Chip/LDB and SSDP both contain N-terminal dimerization domains that constitute the bulk of their structured cores. Here, we report the crystal structures of these dimerization domains, in part aided by DARPin chaperones. We conducted systematic surface scanning by structure-designed mutations, followed by in vitro and in vivo binding assays, to determine conserved surface residues required for binding between Chip/LDB, SSDP, and Pygo-NPFxD. Based on this, and on the 4:2 (SSDP-Chip/LDB) stoichiometry of ChiLS, we derive a highly constrained structural model for this complex, which adopts a rotationally symmetrical SSDP2-LDB2-SSDP2 architecture. Integrity of ChiLS is essential for Pygo binding, and our mutational analysis places the NPFxD pockets on either side of the Chip/LDB dimer, each flanked by an SSDP dimer. The symmetry and multivalency of ChiLS underpin its function as an enhancer module integrating Wnt signals with lineage-specific factors to operate context-dependent transcriptional switches that are pivotal for normal development and cancer.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM/metabolismo , Complexos Multiproteicos/química , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Humanos , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Wnt/genéticaRESUMO
Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII (η6 -arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19 Fâ NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.
Assuntos
Metaloproteínas/metabolismo , Compostos Organometálicos/química , Rutênio/química , Catálise , Flúor , Hidrogenação , Ligantes , Espectroscopia de Ressonância Magnética , Metaloproteínas/química , Estrutura Molecular , Compostos Organometálicos/metabolismo , Rutênio/metabolismoRESUMO
Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. Autophagy receptors target cargo to autophagy by binding ATG8 on autophagosomal membranes. The expansion of the ATG8 family in higher eukaryotes suggests that specific interactions with autophagy receptors facilitate differential cargo handling. However, selective interactors of ATG8 orthologs are unknown. Here we show that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity since cells lacking either protein cannot protect their cytoplasm against Salmonella. LC3C is required for antibacterial autophagy because in its absence the remaining ATG8 orthologs do not support efficient antibacterial autophagy. Structural analysis revealed that the selectivity of NDP52 for LC3C is conferred by a noncanonical LIR, in which lack of an aromatic residue is balanced by LC3C-specific interactions. Our report illustrates that specificity in the interaction between autophagy receptors and autophagy machinery is of functional importance to execute selective autophagy.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Salmonella/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Western Blotting , Cristalografia por Raios X , Citoplasma/metabolismo , Citoplasma/microbiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Salmonella/classificação , Salmonella typhimurium/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including ß-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to ß-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to ß-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein.
Assuntos
Bioensaio/métodos , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Proteína Wnt3A/farmacologia , Regulação para Baixo/efeitos dos fármacos , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Mutação/genética , Domínios PDZ , Peptídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
The proteins MDM2 and MDM4 are key negative regulators of the tumor suppressor protein p53, which are frequently upregulated in cancer cells. They inhibit the transactivation activity of p53 by binding separately or in concert to its transactivation domain. MDM2 is also a ubiquitin ligase that leads to the degradation of p53. Accordingly, MDM2 and MDM4 are important targets for drugs to inhibit their binding to p53. We found from in silico screening and confirmed by experiment that lithocholic acid (LCA) binds to the p53 binding sites of both MDM2 and MDM4 with a fivefold preference for MDM4. LCA is an endogenous steroidal bile acid, variously reported to have both carcinogenic and apoptotic activities. The comparison of LCA effects on apoptosis in HCT116 p53(+/+) vs. p53(-/-) cells shows a predominantly p53-mediated induction of caspase-3/7. The dissociation constants are in the µM region, but only modest inhibition of binding of MDM2 and MDM4 is required to negate their upregulation because they have to compete with transcriptional coactivator p300 for binding to p53. Binding was weakened by structural changes in LCA, and so it may be a natural ligand of MDM2 and MDM4, raising the possibility that MDM proteins may be sensors for specific steroids.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Ácido Litocólico/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatografia de Afinidade , Escherichia coli , Polarização de Fluorescência , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , UltracentrifugaçãoRESUMO
Wnt/ß-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, ß-catenin is targeted continuously for proteasomal degradation by the Axin destruction complex, whose activity is blocked upon Wnt stimulation by Dishevelled, which recruits Axin to the plasma membrane and assembles it into a signalosome. This key event during Wnt signal transduction depends on dynamic head-to-tail polymerization by the DIX domain of Dishevelled. Here, we use rescue assays in Drosophila tissues and functional assays in human cells to show that polymerization-blocking mutations in the DIX domain of Axin disable its effector function in down-regulating Armadillo/ß-catenin and its response to Dishevelled during Wnt signaling. Intriguingly, NMR spectroscopy revealed that the purified DIX domains of the two proteins interact with each other directly through their polymerization interfaces, whereby the same residues mediate both homo- and heterotypic interactions. This result implies that Dishevelled has the potential to act as a "natural" dominant-negative, binding to the polymerization interface of Axin's DIX domain to interfere with its self-assembly, thereby blocking its effector function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biopolímeros/metabolismo , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Fosfoproteínas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Proteína Axina , Proteínas Desgrenhadas , Drosophila , Proteínas de Drosophila/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
p53 maintains genome integrity either by regulating the transcription of genes involved in cell cycle, apoptosis, and DNA repair or by interacting with partner proteins. Here we provide evidence for a direct physical interaction between the tumor suppressors p53 and BRCA2. We found that the transactivation domain of p53 made specific interactions with the C-terminal oligonucleotide/oligosaccharide-binding-fold domains of BRCA2 (BRCA2(CTD)). A second distinct site situated on the p53 DNA-binding domain, bound to a region containing BRC repeats of BRCA2 (BRCA2([BRC1-8])) and may contribute synergistically for high affinity association of intact full-length proteins. Overexpression of BRCA2 and BRCA2(CTD) suppressed the transcriptional activity of p53 with a concomitant reduction in the expression of p53-target genes such as Bax and p21. Consequently, p53-mediated apoptosis was significantly attenuated by BRCA2. The observed physical association of p53 and BRCA2 may have important functional implications in the p53 transactivation-independent suppression of homologous recombination and suggests a possible interregulatory role for both proteins in apoptosis and DNA repair.
Assuntos
Proteína BRCA2/metabolismo , Mapeamento de Interação de Proteínas , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Proteína BRCA2/química , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Regulação para Baixo/genética , Humanos , Cinética , Modelos Biológicos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Transcrição Gênica , Proteína Supressora de Tumor p53/químicaRESUMO
The destabilizing p53 cancer mutation Y220C creates a druggable surface crevice. We developed a strategy exploiting halogen bonding for lead discovery to stabilize the mutant with small molecules. We designed halogen-enriched fragment libraries (HEFLibs) as starting points to complement classical approaches. From screening of HEFLibs and subsequent structure-guided design, we developed substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols as p53-Y220C stabilizers. Crystal structures of their complexes highlight two key features: (i) a central scaffold with a robust binding mode anchored by halogen bonding of an iodine with a main-chain carbonyl and (ii) an acetylene linker, enabling the targeting of an additional subsite in the crevice. The best binders showed induction of apoptosis in a human cancer cell line with homozygous Y220C mutation. Our structural and biophysical data suggest a more widespread applicability of HEFLibs in drug discovery.
Assuntos
Descoberta de Drogas/métodos , Halogênios/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteínas Mutantes , Mutação , Estabilidade Proteica/efeitos dos fármacosRESUMO
Transcriptional silencing through the Polycomb silencing machinery utilizes a "read-write" mechanism involving histone tail modifications. However, nucleation of silencing and long-term stable transmission of the silenced state also requires P-olycomb Repressive Complex 2 (PRC2) accessory proteins, whose molecular role is poorly understood. The Arabidopsis VEL proteins are accessory proteins that interact with PRC2 to nucleate and propagate silencing at the FLOWERING LOCUS C (FLC) locus, enabling early flowering in spring. Here, we report that VEL proteins contain a domain related to an atypical four-helix bundle that engages in spontaneous concentration-dependent head-to-tail polymerization to assemble dynamic biomolecular condensates. Mutations blocking polymerization of this VEL domain prevent Polycomb silencing at FLC. Plant VEL proteins thus facilitate assembly of dynamic multivalent Polycomb complexes required for inheritance of the silenced state.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Polimerização , Inativação Gênica , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Flores/genética , Flores/metabolismoRESUMO
Cilia formation is essential for human life. One of the earliest events in the ciliogenesis program is the recruitment of tau-tubulin kinase 2 (TTBK2) by the centriole distal appendage component CEP164. Due to the lack of high-resolution structural information on this complex, it is unclear how it is affected in human ciliopathies such as nephronophthisis. Furthermore, it is poorly understood if binding to CEP164 influences TTBK2 activities. Here, we present a detailed biochemical, structural, and functional analysis of the CEP164-TTBK2 complex and demonstrate how it is compromised by two ciliopathic mutations in CEP164. Moreover, we also provide insights into how binding to CEP164 is coordinated with TTBK2 activities. Together, our data deepen our understanding of a crucial step in cilia formation and will inform future studies aimed at restoring CEP164 functionality in a debilitating human ciliopathy.
Assuntos
Ciliopatias/genética , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Dicroísmo Circular , Células HEK293 , Humanos , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Estabilidade ProteicaRESUMO
Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K(d) of 1.95 +/- 0.19 microM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM-p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices alpha1 and alpha2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM-p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM-p53 interaction are discussed.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Mitocondriais/química , Multimerização Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/químicaRESUMO
Proteins with intrinsically disordered domains are implicated in a vast range of biological processes, especially in cell signaling and regulation. Having solved the quaternary structure of the folded domains in the tumor suppressor p53 by a multidisciplinary approach, we have now determined the average ensemble structure of the intrinsically disordered N-terminal transactivation domain (TAD) by using residual dipolar couplings (RDCs) from NMR spectroscopy and small-angle x-ray scattering (SAXS). Remarkably, not only were we able to measure RDCs of the isolated TAD, but we were also able to do so for the TAD in both the full-length tetrameric p53 protein and in its complex with a specific DNA response element. We determined the orientation of the TAD ensemble relative to the core domain, found that the TAD was stiffer in the proline-rich region (residues 64-92), which has a tendency to adopt a polyproline II (PPII) structure, and projected the TAD away from the core. We located the TAD in SAXS experiments on a complex between tetrameric p53 and four Taz2 domains that bind tightly to the TAD (residues 1-57) and acted as "reporters." The p53-Taz2 complex was an extended cross-shaped structure. The quality of the SAXS data enabled us to model the disordered termini and the folded domains in the complex with DNA. The core domains enveloped the response element in the center of the molecule, with the Taz2-bound TADs projecting outward from the core.
Assuntos
Ativação Transcricional , Proteína Supressora de Tumor p53/química , Motivos de Aminoácidos , DNA/química , Humanos , Mutação , Fragmentos de Peptídeos , Ligação Proteica , Estrutura Terciária de Proteína , Elementos de Resposta , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The tumor suppressor p53 is mutationally inactivated in approximately 50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in approximately 75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of approximately 150 muM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein-PhiKan083 complex at 1.5-A resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent "druggable" target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.
Assuntos
Genes Supressores de Tumor , Genes p53 , Mutação , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Apoptose , Biofísica/métodos , Calorimetria/métodos , Biologia Computacional , Cristalografia por Raios X/métodos , Análise Mutacional de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Neoplasias/metabolismo , TermodinâmicaRESUMO
The formation of stable 18S U11/U12 di-snRNPs before their association with the pre-mRNA is a characteristic feature of the minor spliceosome. During the spliceosomal assembly, the 18S snRNP binds cooperatively to the introns' 5' splice and branch point site. The molecular basis for this recognition is still unknown. Here, we report the solution structure of the U11-48K CHHC Zn finger, a domain unique to the minor spliceosome. The CHHC Zn-finger structure revealed an unexpected similarity to the TFIIIA domains, with distinct features originating from the type and separation of the zinc-coordinating residues. We show that this domain specifically binds the 5' splice site sequence of U12-type introns when base paired to U11 snRNA in vitro and hence may contribute to the U12 intron recognition. We propose a model in which the U11-48K Zn finger stabilizes U11-5' splice site base pairing and thus plays an important role during the minor spliceosome assembly.
Assuntos
Íntrons , Sítios de Splice de RNA , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/química , Dedos de Zinco , Sequência de Aminoácidos , Pareamento de Bases/fisiologia , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Sítios de Splice de RNA/fisiologia , Estabilidade de RNA/fisiologia , RNA Nuclear Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Soluções , Spliceossomos/química , Spliceossomos/metabolismo , Especificidade por Substrato , Dedos de Zinco/fisiologiaRESUMO
Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic pyrophosphates from metabolic processes. ADP-ribose (ADPR) has been the only known endogenous substrate accepted by NUDT9 so far. The Ca2+ -permeable transient receptor potential melastatin subfamily 2 (TRPM2) channel contains a homologous NUDT9-homology (NUDT9H) domain and is activated by ADPR. Sustained Ca2+ influx via ADPR-activated TRPM2 triggers apoptotic mechanisms. Thus, a precise regulation of cellular ADPR levels by NUDT9 is essential. A detailed characterization of the enzyme-substrate interaction would help to understand the high substrate specificity of NUDT9. Here, we analysed ligand binding to NUDT9 using a variety of biophysical techniques. We identified 2'-deoxy-ADPR as an additional substrate for NUDT9. Similar enzyme kinetics and binding affinities were determined for the two ligands. The high-affinity binding was preserved in NUDT9 containing the mutated NUDIX box derived from the human NUDT9H domain. NMR spectroscopy indicated that ADPR and 2'-deoxy-ADPR bind to the same binding site of NUDT9. Backbone resonance assignment and subsequent molecular docking allowed further characterization of the binding pocket. Substantial conformational changes of NUDT9 upon ligand binding were observed which might allow for the development of NUDT9-based ADPR fluorescence resonance energy transfer sensors that may help with the analysis of ADPR signalling processes in cells in the future.