Human serum albumin (HSA) is the major
protein component of blood plasma and serves as a transporter for
thyroxine and other hydrophobic compounds such as
fatty acids and
bilirubin. We report here a structural characterization of HSA-
thyroxine interactions. Using crystallographic analyses we have identified four binding sites for
thyroxine on HSA distributed in subdomains IIA, IIIA, and IIIB. Mutation of residue R218 within subdomain IIA greatly enhances the affinity for
thyroxine and causes the elevated serum
thyroxine levels associated with
familial dysalbuminemic hyperthyroxinemia (FDH). Structural analysis of two FDH mutants of HSA (R218H and R218P) shows that this effect arises because substitution of R218, which contacts the
hormone bound in subdomain IIA, produces localized conformational changes to relax steric restrictions on
thyroxine binding at this site. We have also found that, although
fatty acid binding competes with
thyroxine at all four sites, it induces conformational changes that create a fifth
hormone-binding site in the cleft between domains I and III, at least 9 A from R218. These structural observations are consistent with binding data showing that HSA retains a high-affinity site for
thyroxine in the presence of excess
fatty acid that is insensitive to FDH mutations.