Flexible loop of beta(2)-glycoprotein I domain V specifically interacts with hydrophobic ligands

beta (2)-Glycoprotein I (beta (2)-GPI), which consists of four complement c ontrol 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-b inding site. We focused on the fifth domain of beta (2)-GPI (Domain V) and examined the interaction of intact Domain V, Domains IV-V, and nicked Domai n 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 D omain V with cleavage in the flexible loop decreases the affinity, indicati ng that the flexible loop region is the binding site of the hydrophobic lig ands. The synthetic peptide corresponding to the loop region was disordered and interacted with bis-ANS, confirming the critical role of the loop regi on. To clarify the nature of the interaction between the loop region and hy drophobic compounds, we prepared the reduced and alkylated Domain V, which was denatured but was assumed to be a collapsed state. Alkylation by iodoac etic acid decreased the interaction of Domain V with bis-ANS, probably beca use the protein net charge was decreased by the six introduced carboxyl gro ups and consequently the electrostatic interactions were decreased. In cont rast, Domain V alkylated by iodoacetamide. therefore retaining a high posit ive net charge, bound bis-ANS more strongly than the intact Domain V. These results suggested that the interaction of Domain V with hydrophobic compou nds through the flexible loop is similar to the binding of hydrophobic comp ounds to the protein folding intermediate.