Conservative mutageneic perturbations of amino acids connecting helix 12 in the 1α,25(OH)2-D3 receptor (VDR) to the ligand cause significant transactivational effects

The positioning of helix 12 activation domain of nuclear receptor proteins is critically important for gene regulation. Perturbations of the helix 12 by larger analogs may alter interactions with transcriptional machinery which might give rise to selectivity. To explore the topology of the ligand binding pocket and how the bound ligand conceivably gives rise to altered transcriptional efficiencies, we have targeted 4 hydrophobic residues which contact the 25-carbon of the ligand, 1α,25(OH)2-vitamin D3, and made a series of 13 mutants. Substitution of a smaller hydrophobic residue was poorly tolerated compared to a larger one for transactivation. The larger amino acids are likely better tolerated by promoting stronger Van der Waals forces with the ligand. Valine-418 mutants demonstrated an extreme example of this observation with mutation to leucine being transactivationally unaffected with alanine being the most affected of all single mutants. V418L resulted in a 1.3-fold increase in EC50 for 1,25-D mediated transactivation whereas V418A resulted in a 53-fold increase when compared to wildtype VDR. Importantly, this difference is not explained by ligand binding data but by differential VDR protease sensitivity implying that V418L-VDR mutation assumes a better conformational interaction surface for coactivator than V418A. Importantly, the V418 location may accommodate larger sidechains and may even enhance the interaction with specific nuclear coactivators.