Thyroxine-binding globulin (TBG) and corticosteroid-binding globulin are unique among non-inhibitory members of the superfamily of serine-proteinase inhibitors (serpins) in undergoing a dramatic increase in stability [stressed-to-relaxed (S→R) transition] after proteolytic cleavage within their exposed reactive-site-loop (RSL) equivalent. This structural rearrangement involves the insertion of the cleaved loop as a new strand into the β-sheet A and is accompanied by a decrease in hormone binding. To define the mechanism that leads to disruption of hormone binding of TBG after proteolytic cleavage, the effect of partial loop deletions and replacements by the α1-proteinase inhibitor homologues of TBG were evaluated. Unexpectedly, deletion of the loop's C-terminus, thought to be important for thyroxine binding, improved the binding affinity over that of normal TBG. Proteolytic cleavage of this variant revealed an intact S→R transition and reduced its binding activity to that of cleaved TBG. In contrast, a chimaera with C-terminal loop extension mimicked the decreased binding affinity of cleaved TBG and had a thermal stability intermediate between that of native and cleaved serpins. This variant was still susceptible to loop cleavage and underwent an S→R transition, yet without changing its binding affinity. Our data exclude a direct involvement of loop residues in thyroxine binding of native TBG. Limited insertion of the RSL into β-sheet A appears to trigger hormone release after proteolytic cleavage. In support of this concept, residues within the hinge region of the TBG loop are phylogenetically highly conserved, suggestive of their physiological role as a functional switch in vivo.
- limited proteolysis
- reactive site
- targeted hormone delivery
- thyroid hormone transport
Abbreviations used: CBG, corticosteroid-binding globulin; HLE, human leucocyte elastase; Ka, affinity constant [thyroxine (T4)-binding]; α1-PI, α1-proteinase inhibitor; RSL, reactive-site loop; s, β-strand (e.g. s1C); Sf9, Spodoptera frugiperda 9; S→R transition, stressed-to-relaxed transition; serpin(s), serine proteinase inhibitor(s); T3, tri-iodothyronine; TBG, thyroxine-binding globulin; residues are numbered as described by Schechter and Berger [ 1], where P1—P1′ is the scissile bond, P2, P3, etc. are residues in the N-terminal direction and P1′, P2′, etc. are residues in the C-terminal direction; for the non-inhibitory TBG, the numbers correspond to the equivalent positions of α1-PI based on the alignment of the conserved regions preceding and following the RSL respectively; the denotation of serpin secondary-structural elements and their assignment to the TBG sequence are as described in [ 2].
- The Biochemical Society, London ©2002