Linkage between the hormone binding site and the reactive center loop of thyroxine binding globulin.
Thyroxine binding globulin (TBG) is the major carrier of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in plasma. TBG is member of the serpin family of proteins although it has no proteinase inhibitory activity. In this study we show that TBG has properties typical of a metastable serpin and provide evidence that occupancy of the hormone binding site alters the conformation of the reactive center loop. After reactive center loop cleavage by endoproteinase Asp-N or neutrophil elastase the protein became more stable to guanidine hydrochloride denaturation compared to the native protein, as a result of loop insertion. In addition, incubation of the native protein with a reactive center loop peptide, caused a change in mobility on a native gel. This is consistent with the idea that thyroxine binding globulin is able to form a binary complex with the peptide as a result of beta-sheet A expansion. To assess the effect of cleavage and loop insertion on the hormone binding site we used the specific binding of a fluorophore, 1,8-anilinonaphthalene sulfonic acid (ANS). Loop insertion itself had no effect on ANS affinity, but cleavage with elastase at the P4'-P5' bond caused a reduction in affinity, presumably because this cleavage site is located within the hormone binding site. These data support the concept that cleavage of TBG by proteinases released in inflammation is a mechanism to deliver thyroid hormones to target tissues. A linkage between the occupancy state of the hormone binding site and the conformation of the reactive center loop was indicated by the observation that binding of T3 to native TBG reduced proteolytic susceptibility by both endoproteinase Asp-N and elastase.
Suda
S A
SA
Department of Oral Medicine and Diagnostic Sciences, Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago,  60612, USA.
Gettins
P G
PG
Patston
P A
PA
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D006728:Hormones / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D013975:Thyroxine-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D006728:Hormones; D013975:Thyroxine-Binding Proteins; D010447:Peptide Hydrolases