Roles of the Structure and Orientation of Ligands and Ligand Mimics inside the Ligand-Binding Pocket of the Vitamin D-Binding Protein

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Abstract
1alpha,25-Dihydroxyvitamin D3, the vitamin D hormone, manifests its diverse biological properties by specifically binding to the vitamin D sterol-binding pockets of vitamin D-binding protein (DBP) and vitamin D receptor. In the past, several affinity, photoaffinity, and chemical modification studies have been carried out to probe the vitamin D sterol-binding pocket of DBP and to evaluate the relationship between the structure of this pocket and the functions of the protein. In the present study, we examined the steric requirements inside this pocket by considering conformational structures of various bromoacetate derivatives of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 and their abilities to covalently and specifically modify this pocket. We observed that, although 25-hydroxyvitamin D3 3beta-bromoacetate (25-OH-D3-3-BE), 1alpha,25-dihydroxyvitamin D3 3beta-bromoacetate [1alpha,25(OH)2D3-3-BE], 1alpha,25-dihydroxyvitamin D3 1alpha-bromoacetate [1alpha,25(OH)2D3-1-BE], and 1alpha,25-dihydroxyvitamin D3 1alpha,3beta-dibromoacetate [1alpha,25(OH)2D3-1,3-di-BE] bound DBP in a specific manner, only [3H]-25-OH-D3-3-BE and [3H]-1alpha,25(OH)2D3-3-BE affinity labeled the protein. BNPS-skatole cleavages of [3H]-25-OH-D3-3-BE- and 3H-1alpha,25(OH)2D3-3-BE-labeled DBP samples produced the same labeled peptide (N-terminal), demonstrating the specificity of labeling by these analogs. Energy-minimized conformational structures of these bromoacetate derivatives indicated significant changes in the A-ring conformations of these analogs. These structural changes were invoked to explain the inability of [3H]-1alpha,25(OH)2D3-1-BE and [3H]-1alpha,25(OH)2D3-1,3-di-BE to affinity label DBP. Overall, these studies suggested that the vitamin D sterol-binding pocket in DBP is sterically quite restrictive. This information could be potentially important in designing future vitamin D-based drugs for several diseases.