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 alpha,25-dihydroxyvitamin D3

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, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-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 beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). 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 beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-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 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.     

2-Methylene 19-nor-25-dehydro-1alpha-hydroxyvitamin D3 26,23-lactones: synthesis, biological activities and molecular basis of passive antagonism

To investigate the molecular mechanism of vitamin D receptor (VDR) antagonists having no structurally bulky group interfering with helix 12 of the ligand-binding domain of the VDR, we have synthesized four diastereomers at C(20) and C(23) of 19-nor-1alpha-hydroxyvitamin D(3) 25-methylene-26,23-lactone bearing a 2MD-type A-ring. All four analogs showed significant VDR affinity. Transactivation was tested by using Cos7 cells and HEK293 cells. In both types of cells, LAC67a showed little transactivation potency and inhibited the activation induced by the natural hormone concentration-dependently, indicating that LAC67a works as an antagonist for the VDR in these cells. LAC67b, LAC82a and LAC82b similarly acted as VDR antagonists in Cos7 cells, but in HEK293 cells they behaved as potent VDR agonists. Docking of four lactones into the VDR-LBD, followed by structural analysis, demonstrated that each lactone lacks the hydrophobic interaction with helix12 necessary for maintaining the active conformation of the VDR, indicating that these lactones are passive-type antagonists. Furthermore, each docking structure explained the characteristic transactivation profiles of the four lactones. On the basis of our present findings, we suggest that the ligand acts as an agonist if there are appropriate coactivators in the cells to bind to the looser VDR-ligand complex, and as an antagonist if there are no such appropriate coactivators. The molecular basis of the passive antagonism is discussed in detail.     

A GX2GX3G motif facilitates acyl chain sequestration by Saccharomyces cerevisiae acyl carrier protein

Saccharomyces cerevisiae acyl carrier protein (ScACP) is a component of the large fungal fatty acid synthase I (FAS I) complex. ScACP comprises two subdomains: a conserved ACP domain that shares extensive structural homology with other ACPs and a unique structural domain. Unlike the metazoan type I ACP that does not sequester the acyl chain, ScACP can partially sequester the growing acyl chain within its hydrophobic core by a mechanism that remains elusive. Our studies on the acyl-ScACP intermediates disclose a unique ¹⁸⁸GX₂GX₃G¹⁹⁵ sequence in helix II important for ACP function. Complete loss of sequestration was observed upon mutation of the three glycines in this sequence to valine (G188V/G191V/G195V), while G191V and G188V/G191V double mutants displayed a faster rate of acyl chain hydrolysis. Likewise, mutation of Thr216 to Ala altered the size of the hydrophobic cavity, resulting in loss of C₁₂- chain sequestration. Combining NMR studies with insights from the crystal structure, we show that three glycines in helix II and a threonine in helix IV favor conformational change, which in turn generate space for acyl chain sequestration. Furthermore, we identified the primary hydrophobic cavity of ScACP, present between the carboxyl end of helix II and IV. The opening of the cavity lies between the second and third turns of helix II and loop II. Overall, the study highlights a novel role of the GX₂GX₃G motif in regulating acyl chain sequestration, vital for ScACP function.