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 1
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- 作者：Narasimha Swamy ; Wenrong Xu ; Nancy Paz ; Jui-Cheng Hsieh ; Mark R. Haussler ; George J. Maalouf ; Scott C. Mohr ; and Rahul Ray
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：October 10, 2000
- 年：2000
- 卷：39
- 期：40
- 页码：12162 - 12171
- 全文大小：303K
- 年卷期：v.39,no.40(October 10, 2000)
- ISSN：1520-4995
文摘
We have combined molecular modeling and classical structure-function techniques to definethe interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) andits natural ligand, 1
,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The affinity analogue 1,25-(OH)2D3-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolishedthis affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines inthe VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separatelycreating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating thecrucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBDsequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 andW286 in the presumed 
-hairpin of the molecule, thereby identifying it as the region contacting the A-ringof 1,25-(OH)2D3. From the multiple sequence alignment, we developed a homologous extension modelof the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the -conformationof the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. Themodel predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at thesite 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 1,25-(OH)2D3-binding motifs in the ligand-binding pocket of VDR.
,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The affinity analogue 1,25-(OH)2D3-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolishedthis affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines inthe VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separatelycreating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating thecrucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBDsequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 andW286 in the presumed -hairpin of the molecule, thereby identifying it as the region contacting the A-ringof 1,25-(OH)2D3. From the multiple sequence alignment, we developed a homologous extension modelof the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the -conformationof the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. Themodel predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at thesite 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 1,25-(OH)2D3-binding motifs in the ligand-binding pocket of VDR.