Flexible Loop of β2-Glycoprotein I Domain V Specifically Interacts with Hydrophobic Ligands

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Abstract
β2-Glycoprotein I (β2-GPI), which consists of four complement control protein modules and a distinctly folded fifth C-terminal domain, is an essential cofactor for the binding to phospholipids of anti-cardiolipin antibodies, isolated from patients with anti-phospholipid antibody syndrome, and its fifth domain has attracted attention as a specific phospholipid-binding site. We focused on the fifth domain of β2-GPI (Domain V) and examined the interaction of intact Domain V, Domains IV−V, and nicked Domain V with various hydrophobic ligands, as a model molecule of phospholipid. We found that electrostatic and hydrophobic interactions are important for Domain V binding to the ligand molecules. We also found that, while Domain IV has no significant effect on the interactions with ligands, the nicked Domain V with cleavage in the flexible loop decreases the affinity, indicating that the flexible loop region is the binding site of the hydrophobic ligands. The synthetic peptide corresponding to the loop region was disordered and interacted with bis-ANS, confirming the critical role of the loop region. To clarify the nature of the interaction between the loop region and hydrophobic compounds, we prepared the reduced and alkylated Domain V, which was denatured but was assumed to be a collapsed state. Alkylation by iodoacetic acid decreased the interaction of Domain V with bis-ANS, probably because the protein net charge was decreased by the six introduced carboxyl groups and consequently the electrostatic interactions were decreased. In contrast, Domain V alkylated by iodoacetamide, therefore retaining a high positive net charge, bound bis-ANS more strongly than the intact Domain V. These results suggested that the interaction of Domain V with hydrophobic compounds through the flexible loop is similar to the binding of hydrophobic compounds to the protein folding intermediate.