RESUMO
Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.
Assuntos
Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , UbiquitinasRESUMO
Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.
Assuntos
Proteínas de Bactérias/metabolismo , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Técnicas Biossensoriais , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ligantes , Família Multigênica , Mutação , Naftalenos/metabolismo , Naftoquinonas/metabolismo , Piranos/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/genética , Tetraciclina/farmacologiaRESUMO
The SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase family was discovered through genetic requirements for cell cycle progression in budding yeast. In these multisubunit enzymes, an invariant core complex, composed of the Skp1 linker protein, the Cdc53/Cul1 scaffold protein and the Rbx1/Roc1/Hrt1 RING domain protein, engages one of a suite of substrate adaptors called F-box proteins that in turn recruit substrates for ubiquitination by an associated E2 enzyme. The cullin-RING domain-adaptor architecture has diversified through evolution, such that in total many hundreds of distinct SCF and SCF-like complexes enable degradation of myriad substrates. Substrate recognition by adaptors often depends on posttranslational modification of the substrate, which thus places substrate stability under dynamic regulation by intracellular signaling events. SCF complexes control cell proliferation through degradation of critical regulators such as cyclins, CDK inhibitors and transcription factors. A plethora of other processes in development and disease are controlled by other SCF-like complexes, including those based on Cul2-SOCS-box adaptor protein and Cul3-BTB domain adaptor protein combinations. Recent structural insights into SCF-like complexes have begun to illuminate aspects of substrate recognition and catalytic reaction mechanisms.
Assuntos
Proteínas Culina/fisiologia , Ubiquitina/fisiologia , Sequência de Aminoácidos , Proteínas de Arabidopsis/fisiologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Saccharomycetales/enzimologia , Saccharomycetales/fisiologia , Alinhamento de Sequência , Ubiquitinas/fisiologiaRESUMO
The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.