Mutational Analysis and Engineering of the Human Vitamin D Receptor to Bind a Novel Small Molecule

Hilda Castillo
Georgia Institute of Technology, School of Chemistry & Biochemistry, Atlanta, GA 30332 USA

Abstract:
Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of genes involved in biologically important processes. The diversity of ligand binding among NRs makes NRs attractive candidates for protein engineering, such that these receptors can be engineered to bind novel small molecules for several applications. One application is the creation of potential molecular switches, tools that can be used for controlling gene expression.

The human vitamin D receptor (hVDR) was engineered using two approaches. In the first approach, structural analysis and in silico modeling allowed the rational design of two libraries using randomized codons at selected positions. The second approach involved random mutagenesis, using error-prone PCR to create libraries of variants. All of the hVDR variants obtained were analyzed using chemical complementation, a genetic selection system in which the survival of yeast is linked to the activation of a NR by an agonist.

A variant, hVDRC410Y that is activated by a known hVDR ligand with enhanced sensitivity in comparison to the wild-type hVDR, and is also activated by a novel small molecule that does not activate the wild-type hVDR was discovered. The effects of residue C410 on ligand specificity and activation are surprising, as this residue does not line the hVDR’s ligand binding pocket and as a result does not make direct contacts with the receptor’s natural ligand.