Characterization of the thyroxine-binding site of thyroxine-binding globulin by site-directed mutagenesis

The principal transport protein for T₄ in human blood, thyroxine-binding globulin (TBG), binds T₄ with an exceptionally high affinity (K(a) = 10¹⁰ M^-1). Its homology to the superfamily of the serpins has recently been used in the design of chimeric proteins, providing experimental evidence that an eight-stranded β-barrel domain encompasses the ligand-binding site. We have now characterized the T₄ binding site by site-directed mutagenesis. Sequence alignment of TBG from several species revealed a phylogenetically highly conserved stretch of amino acids comprising strands 2B and 3B of the β-barrel motif. Mutations within this region (Val²²⁸Glu, Cys²³⁴Trp, Thr²³⁵Trp, Thr²³⁵Gln, Lys²⁵³Ala, and Lys²⁵³Asp), designed to impose steric hindrance or restriction of its mobility, had no significant influence on T₄ binding. However, binding affinity was 20-fold reduced by introduction of an N-linked glycosylation site at the turn between strands 2B and 3B (Leu²⁴⁶Thr) without compromising the proper folding of this mutant as assessed by immunological methods. In most other serpins, this glycosylation site is highly conserved and has been shown to be crucial for cortisol binding of corticosteroid-binding globulin, the only other member of the serpins with a transport function. The ligand-binding site could thus be located to a highly aromatic environment deep within the β-barrel. The importance of the binding site's aromatic character was investigated by exchanging phenylalanines with alanines. Indeed, these experiments revealed that substitution of Phe²⁴⁹ in the middle of strand 3B completely abolished T₄ binding, while the substitution of several other phenylalanines had no effect.