RESUMO
Overexpression of multiple copies in T-cell lymphoma-1 (MCT-1) oncogene accompanies malignant phenotypic changes in human lymphoma cells. Specific disruption of MCT-1 results in reduced tumorigenesis, suggesting a potential for MCT-1-targeted therapeutic strategy. MCT-1 is known as a cap-binding protein and has a putative RNA-binding motif, the PUA-domain, at its C-terminus. We determined the crystal structure of apo MCT-1 at 1.7 Å resolution using the surface entropy reduction method. Notwithstanding limited sequence identity to its homologs, the C-terminus of MCT-1 adopted a typical PUA-domain fold that includes secondary structural elements essential for RNA recognition. The surface of the N-terminal domain contained positively charged patches that are predicted to contribute to RNA-binding.
Assuntos
Proteínas de Ciclo Celular/química , Mutação , Proteínas Oncogênicas/química , Proteínas Recombinantes de Fusão/química , Alanina/química , Alanina/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Fenômenos Biofísicos , Proteínas de Ciclo Celular/genética , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Cristalografia/métodos , DNA Complementar/química , DNA Complementar/genética , Entropia , Escherichia coli/química , Escherichia coli/genética , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteínas Oncogênicas/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico , Eletricidade Estática , TemperaturaRESUMO
We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.