RESUMO
TGIF1 is a multifunctional protein that represses TGF-ß-activated transcription by interacting with Smad2-Smad4 complexes. We found that the complex structure of TGIF1-HD bound to the TGACA motif revealed a combined binding mode that involves the HD core and the major groove, on the one hand, and the amino-terminal (N-term) arm and the minor groove of the DNA, on the other. We also show that TGIF1-HD interacts with the MH1 domain of Smad proteins, thereby indicating that TGIF1-HD is also a protein-binding domain. Moreover, the formation of the HD-MH1 complex partially hinders the DNA-binding site of the complex, preventing the efficient interaction of TGIF1-HD with DNA. We propose that the binding of the TGIF1 C-term to the Smad2-MH2 domain brings both the HD and MH1 domain into close proximity. This local proximity facilitates the interaction of these DNA-binding domains, thus strengthening the formation of the protein complex versus DNA binding. Once the protein complex has been formed, the TGIF1-Smad system would be released from promoters/enhancers, thereby illustrating one of the mechanisms used by TGIF1 to exert its function as an active repressor of Smad-induced TGF-ß signaling.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Proteínas Repressoras/química , Proteína Smad2/química , Proteína Smad4/química , Fator de Crescimento Transformador beta/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Motivos de Nucleotídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Herein, the stereostructure of the aromatic heptaene macrolide (AHM) antifungal antibiotic candicidin A3 (syn. ascosin A3, levorin A3) has been established upon the 2D NMR studies, consisting of DQF-COSY, TOCSY, ROESY, HSQC and HMBC experiments, as well as upon extensive molecular dynamics simulations. The geometry of the heptaenic chromophore was defined as: (22E, 24E, 26Z, 28Z, 30E, 32E, 34E). The previously unreported absolute configuration of the chiral centres of candicidin A3 was established as: (3R, 9R, 11S, 13S, 15R, 17S, 18R, 19S, 21R, 36S, 37R, 38S, 40S, 41S).