Loop variants of the serpin thyroxine-binding globulin: implications for hormone release upon limited proteolysis.
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 beta-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 alpha(1)-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 beta-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.
Grasberger
Helmut
H
Howard Hughes Medical Institute, University of Chicago, 5841 S. Maryland Ave, Chicago, IL 60637, USA.
Golcher
Henriette M B
HM
Fingerhut
Anja
A
Janssen
Onno E
OE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004355:Drug Stability; D014644:Genetic Variation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013974:Thyroxine / Q000378:metabolism; D013975:Thyroxine-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013975:Thyroxine-Binding Proteins; D019272:Leukocyte Elastase; D013974:Thyroxine