RESUMEN
Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.
Asunto(s)
Uniones Adherentes/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Polaridad Celular/fisiología , Drosophila , Proteínas de Drosophila/genética , Epitelio/crecimiento & desarrollo , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Fosforilación , Unión Proteica/genética , Estructura Terciaria de ProteínaRESUMEN
The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin- and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.
Asunto(s)
Enfermedad de Machado-Joseph/enzimología , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Ataxina-3 , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , Dipéptidos , Escherichia coli , Leucina/análogos & derivados , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares , Estructura Terciaria de Proteína , Proteínas Represoras , Especificidad por SustratoRESUMEN
The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)(2) domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3' flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.
Asunto(s)
ADN de Archaea/química , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Aeropyrum/genética , Aeropyrum/metabolismo , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Reparación del ADN , ADN de Archaea/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Sustancias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
BACKGROUND: Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase. Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active. The molecular mechanisms underlying this dual regulation of separase by securin are unknown. RESULTS: We show that, in budding yeast, securin supports separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside. Securin also ensures that separase gains full proteolytic activity in anaphase. We also show that securin, while present, directly inhibits the proteolytic activity of separase. Securin prevents the binding of separase to its substrates. It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus. CONCLUSIONS: Securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset.