The high affinity ligand binding conformation of the nuclear 1,25-dihydroxyvitamin D3 receptor is functionally linked to the transactivation domain 2 (AF-2).

The nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), VDR, is a transcription factor that mediates all genomic actions of the hormone. The activation of VDR by ligand induces a conformational change within its ligand binding domain (LBD). Due to the lack of a crystal structure analysis, biochemical methods have to be applied in order to investigate the details of this receptor-ligand interaction. The limited protease digestion assay can be used as a tool for the determination of a functional dissociation constant (K(df)) of VDR with any potential ligand. This method provided with the natural hormone VD two protease-resistant fragments of the VDR LBD and with the 20-epi conformation of VD, known as MC1288, even an additional fragment of intermediate size. These fragments were interpreted as different receptor conformations and their decreasing size was found to be associated with decreasing ligand binding affinity. A critical amino acid for VDR's high ligand binding conformation has been identified by C-terminal receptor truncations and point mutations as phenylalanine 422. This amino acid appears to directly contact the ligand and belongs to the ligand-inducible activation function-2 (AF-2) domain. Moreover, functional assays supported the observation that high affinity ligand binding is directly linked to transactivation function.