To repress gene transcription, the unliganded nuclear receptor (NR) recruits the N-CoR and SMRT corepressors via its direct association with the conserved motif within bipartite NR-interaction domains (IDs) of corepressors. We recently reported that SMRT is directly involved in the VDR-mediated repression via an ID1-specific interaction with the VDR. Here we show that removal of helix 12 from VDR (VDR triangle AF2) converts it to a more potent repressor through additional interaction between the VDR and SMRT-ID2 in yeast and mammalian systems. These data suggest that the VDR helix 12 actively regulates the ID1 preference of the VDR by inhibiting ID2-VDR association. Using the one- Plus two-hybrid system, we identified specific residues within the extended helix motif of SMRT-ID2 that are required for VDR triangle AF2 binding. Analyses of these mutants also revealed the specific residues of SMRT-ID2 generally required for optimal NR binding as well as those involved in preferential interaction with specific NRs. (c) 2008 Elsevier Inc. All rights reserved.