RESUMO
The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. By affinity chromatography, native gel electrophoresis and fluorescence spectroscopy, we show the calcium-dependent binding of SRY to CaM. Binding occurs via the HMG box and an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and we propose that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors.
Assuntos
Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Autorradiografia , Sequência de Bases , Sítios de Ligação , Cálcio/farmacologia , Cromatografia de Afinidade , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Radioisótopos de Fósforo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sitios de Sequências Rotuladas , Proteína da Região Y Determinante do Sexo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Testículo/metabolismoRESUMO
The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.
