RESUMO
Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5â Å resolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination.
Assuntos
Proteínas Culina/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte/química , Cristalografia por Raios X , Proteínas Culina/genética , Proteínas Culina/ultraestrutura , Humanos , Microscopia de Força Atômica , Modelos Químicos , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestrutura , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteínas Repressoras/genética , Proteínas Repressoras/ultraestrutura , Especificidade por Substrato/genética , Ubiquitinação/genética , Regulação para Cima/genéticaRESUMO
Bach2 is a transcriptional repressor that is expressed during specific stages of B-cell development and in neuronal cells. It plays a critical role in modulating class-switch recombination during the differentiation of mature B cells to antibody-secreting plasma cells and it is also an important regulator of apoptotic responses to oxidative stress. Bach2 has been implicated both as an oncogene and as a tumour suppressor in human malignancy. The interaction of Bach2 with its target genes is mediated via its basic leucine-zipper region, whereas the N-terminal POZ domain recruits transcriptional co-repressors and class II histone deacetylases. Here, the crystal structure of the human Bach2 POZ domain is reported at 2.1 Å resolution. The Bach2 POZ-domain dimer resembles the POZ-domain dimers of the POZ zinc finger transcription factors and dimerization is independent of an N-terminal region that has previously been implicated in the dimerization of the POZ basic leucine-zipper protein Bach1. The Bach2 POZ domain crystallized in two forms which differed by the presence of an intersubunit disulfide bond. The intersubunit disulfide bond is present both in bacterially expressed Bach2 POZ domain in solution and in protein expressed in transfected eukaryotic cells. These crystal structures will be relevant for understanding the regulation of Bach2 in response to oxidative stress and for the design of therapeutics that target the Bach2 POZ domain in human malignancy.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , Dissulfetos/química , Multimerização Proteica , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células COS , Chlorocebus aethiops , Dissulfetos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
BCL6 is a transcriptional repressor that is overexpressed in diffuse large B-cell lymphoma and follicular lymphoma. The N-terminal POZ domain of BCL6 interacts with transcriptional corepressors and targeting these associations is a promising therapeutic strategy. Previous structural studies of the BCL6 POZ domain have used a mutant form because of the low solubility of the wild-type recombinant protein. A method for the purification and crystallization of the wild-type BCL6 POZ domain is described and the crystal structure to 2.1 A resolution is reported. This will be relevant for the design of therapeutics that target BCL6 POZ-domain interaction interfaces.