Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein.
Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.
Swamy
Narasimha
N
Bioorganic Chemistry & Structural Biology, Section in Endocrinology, Diabetes and Metabolism, Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Head
James F
JF
Weitz
Daniel
D
Ray
Rahul
R
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D002112:Calcifediol / Q000378:metabolism*; D002117:Calcitriol / Q000378:metabolism*; D002846:Chromatography, Affinity; D018360:Crystallography, X-Ray; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism; D014809:Vitamin D-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D014809:Vitamin D-Binding Protein; D014357:Trypsin; D002117:Calcitriol; D002112:Calcifediol