Probing the Vitamin D Sterol-Binding Pocket of Human Vitamin D-Binding Protein with Bromoacetate Affinity Labeling Reagents Containing the Affinity Probe at C-3, C-6, C-11, and C-19 Positions of Parent Vitamin D Sterols

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D3 and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D3 [25-OH-D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)2-D3-11-BE, 25-OH-D3-3-BE, and 25-OH-D3-6-BE, respectively, compared to that seen with 25-OH-D3, while there was no significant difference in the DBP-binding affinity of 25-OH-D3-19-BE and 25-OH-D3. Surprisingly, [14C]25-OH-D3-11-BE and [14C]1,25(OH)2-D3-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, [14C]25-OH-D3-6-BE and [14C]25-OH-D3-3-BE specifically labeled DBP. BNPS–skatole digestion of DBP labeled with [14C]25-OH-D3-3-BE or [14C]25-OH-D3-6-BE produced two peptides (Mr 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1–203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for “mapping” the vitamin D sterol-binding pocket and developing a functional model.