Title:
Mutational analysis and engineering of the human vitamin D receptor to bind and activate in response to a novel small molecule ligand

dc.contributor.advisor Azizi, Bahareh
dc.contributor.advisor Doyle, Donald F.
dc.contributor.advisor Bommarius, Andreas S.
dc.contributor.advisor Williams, Loren D.
dc.contributor.author Castillo, Hilda S. en_US
dc.contributor.committeeMember Hud, Nicholas
dc.contributor.committeeMember May, Sheldon
dc.contributor.department Chemistry and Biochemistry en_US
dc.date.accessioned 2011-07-06T16:25:05Z
dc.date.available 2011-07-06T16:25:05Z
dc.date.issued 2011-01-22 en_US
dc.description.abstract Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of genes involved in all physiological activities. Disruption in NR function (e.g. mutations) can lead to a variety of diseases; making these receptors important targets for drug discovery. The ability to bind a broad range of 'drug-like' molecules also make these receptors attractive candidates for protein engineering, such that they can be engineered to bind novel small molecule ligands, for several applications. One application is the creation of potential molecular switches, tools that can be used for controlling gene expression. Gaining knowledge of specific molecular interactions that occur between a receptor and its ligand is of interest, as they contribute towards the activation or repression of target genes. The focus of this work has been to investigate the structural and functional relationships between the human vitamin D receptor (hVDR) and its ligands. To date, mutational assessments of the hVDR have focused on alanine scanning and residues typically lining the ligand binding pocket (LBP)that are involved in direct interactions with the ligand. A comprehensive analysis of the tolerance of these residues in the binding and activation of the receptor by its ligands has not been performed. Furthermore, residues not in contact with the ligand or that do not line the LBP may also play an important role in determining the activation profiles observed for NRs, and therefore need to be explored further. In order to engineer and use the hVDR in chemical complementation, a genetic selection system in which the survival of yeast is linked to the activation of a NR by an agonist, the hVDR gene was isolated from cDNA. To gain insight into how chemical and physical changes within the ligand binding domain (LBD) affect receptor-ligand interactions, libraries of hVDR variants exploring the role and tolerance of hVDR residues were created. To develop a comprehensive mutational analysis while also engineering the hVDR to bind a novel small molecule ligand, a rational and a random mutagenic approach were used to create the libraries. A variant, hVDRC410Y, that displayed enhanced activity with lithocholic acid (LCA), a known hVDR ligand, and novel activation with cholecalciferol (chole), a precursor of the hVDR's natural ligand known not to activate the wild-type hVDR, was discovered. The presence of a tyrosine at the C410 position resulting in novel activation profiles with both LCA and chole, and the fact that this residue does not line the hVDR's LBP led to interest in determining whether a physical or chemical property of the residue was responsible for the observed activity. When residue C410 was further assessed for its tolerance to varying amino acids, the results indicated that bulkiness at this end of the pocket is important for activation with these ligands. Both LCA and chole have reduced molecular volumes compared to the natural ligand, 1alpha, 25(OH)2D3. As a result, increased bulkiness at the C410 position may contribute additional molecular interactions between the receptor and ligands. Results obtained throughout this work suggest that the end of the hVDR's LBP consisting of two ligand anchoring residues, H305 and H397, and residue C410 tolerates structural variations, as numerous variants with mutations at these positions displayed enhanced activity. The receptor contains two tyrosines, Y143 and Y147, which were targeted for mutagenesis in one of the rationally designed libraries, located at the exact opposite end of the pocket. In an effort to gain further insight into the role of these residues at the other end of the LBP, mutagenesis assessing the tolerance of tyrosines 143 and 147 was performed. Overall, most changes at these positions proved to be detrimental to the function of the receptor supporting the hypothesis that this end of the LBP is less tolerant of structural changes, compared to the opposite end consisting of residues H305, H397 and C410. Overall, a better understanding of the structural and functional relationships between the human vitamin D receptor (hVDR) and its ligands was achieved. The effects of residue C410 on specificity and activation with the different ligands studied were unforeseen, as this residue does not line the receptor's ligand binding pocket (LBP). However, they serve as an example of the significant impact distant residues can have on receptor activation and also emphasize the important role physical properties of residues, such as volume, can play for specific ends of the LBP compared to chemical properties. en_US
dc.description.degree Ph.D. en_US
dc.identifier.uri http://hdl.handle.net/1853/39502
dc.publisher Georgia Institute of Technology en_US
dc.subject Mutagenesis en_US
dc.subject Protein engineering en_US
dc.subject Nuclear receptor en_US
dc.subject Vitamin D receptor en_US
dc.subject Lithocholic acid en_US
dc.subject Cholecalciferol en_US
dc.subject.lcsh Ligands (Biochemistry)
dc.subject.lcsh Vitamin D in the body
dc.subject.lcsh Nuclear receptors (Biochemistry)
dc.title Mutational analysis and engineering of the human vitamin D receptor to bind and activate in response to a novel small molecule ligand en_US
dc.type Text
dc.type.genre Dissertation
dspace.entity.type Publication
local.contributor.advisor Williams, Loren D.
local.contributor.advisor Bommarius, Andreas S.
local.contributor.corporatename School of Chemistry and Biochemistry
local.contributor.corporatename College of Sciences
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relation.isAdvisorOfPublication faf2f937-2845-4a43-af4c-3d34a45ece32
relation.isOrgUnitOfPublication f1725b93-3ab8-4c47-a4c3-3596c03d6f1e
relation.isOrgUnitOfPublication 85042be6-2d68-4e07-b384-e1f908fae48a
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