Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein

Abstract

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 Å crystal, primitive orthorhombic with unit cell dimensions a=80.2 Å, b=87.3 Å, and c=159.6 Å, P2 12 12 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.