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MH1 Domain of SMAD4 Binds N-terminal Residues of the Homeodomain of Hoxc9

【摘要】:正Smad family proteins mediate signaling initiated by bone morphogenetic proteins(BMPs).Upon BMP stimulation,the Smads such as Smad4 can interact directly with Hox proteins and suppress their DNA-binding activity.Although the interaction between the MAD-homology 1(MH1)domain of Smad4 and Hox was found to regulate the transcription activity of Hox proteins,the molecular mechanism is not well characterized and direct contact residues remain to be elucidated.In the present study,the interaction between the recombinant homeodomain(HD)of Hoxc9 and MH1 domain of Smad4 was investigated with the use of the GST pull-down assay,surface Plasmon resonance(SPR)analysis as well as multidimensional nuclear magnetic resonance(NMR)techniques.The Hoxc9-HD was precipitated with the GST-fused Smad4-MH1 but not with GST alone,demonstrating a direct interaction between Hoxc9-HD and Smad4-MH1 in vitro.SPR measurement further confirmed a moderately strong interaction(K_d≈400nM)between these two domains.Moreover,NMR titration experiments showed that a strong and specific binding occurred between Smad4-MH1 and Hoxc9-HD.NMR triple-resonance experiments and backbone assignments revealed that the N-terminal arm of Hoxc9-HD,spanning the positive-charged DNA-binding segment of Arg 190-Arg196, was intimately involved in the interaction with Smad4-MH1.Ala-substitutions of Arg190-Arg196 led to the loss of interaction between Hoxc9-MH1 and Smad4-MH1 in both GST-pull down assay and SPR analysis;further provided functional evidence for the critical role of this positive-charged region in binding to Smad4-MH1.This suggested that Smad4-MH1 could occupy one of the DNA binding sites of Hoxc9 and consequently inhibits its transcription activity.The above results are in good agreement and yield the first insight into the interaction between the homeodomain of Hox proteins and the conserved MH1 domain of Smad family proteins.

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