Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1α,25-dihydroxyvitamin D3

N. Swamy, W. Xu, N. Paz, J. C. Hsieh, M. R. Haussler, G. J. Maalouf, S. C. Mohr, R. Ray

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3]. The affinity analogue 1α,25-(OH)2D3-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed β-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1α,25-(OH)2D3. From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single β hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the α-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the 'activation' helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1α,25-(OH)2D3-binding motifs in the ligand-binding pocket of VDR.

Original languageEnglish (US)
Pages (from-to)12162-12171
Number of pages10
JournalBiochemistry
Volume39
Issue number40
DOIs
StatePublished - Oct 10 2000

ASJC Scopus subject areas

  • Biochemistry

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