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 D(3) 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 D(3) ¿25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ¿1,25(OH)(2)D(3) 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)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ¿(14)C25-OH-D(3)-11-BE and ¿(14)C1, 25(OH)(2)-D(3)-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, ¿(14)C25-OH-D(3)-6-BE and ¿(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ¿(14)C25-OH-D(3)-3-BE or ¿(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 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.
Swamy
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Vitamin D Laboratory, Department of Medicine, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118, USA.
Addo
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Vskokovic
M R
MR
Ray
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R
D000085:Acetates / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002112:Calcifediol / Q000031:analogs & derivatives / Q000378:metabolism*; D002250:Carbon Radioisotopes; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D012862:Skatole / Q000031:analogs & derivatives / Q000378:metabolism; D014809:Vitamin D-Binding Protein / Q000737:chemistry*
D000085:Acetates; D000345:Affinity Labels; D002250:Carbon Radioisotopes; D014809:Vitamin D-Binding Protein; C005904:BNPS-skatole; C016274:bromoacetate; D012862:Skatole; D002112:Calcifediol