{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T16:15:21Z","timestamp":1759940121264},"reference-count":42,"publisher":"The Endocrine Society","issue":"11","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[1999,11,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The principal transport protein for T4 in human blood, thyroxine-binding globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 1010m\u22121). 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 \u03b2-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 \u03b2-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\u03b2 -barrel. The importance of the binding site\u2019s 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.<\/jats:p>","DOI":"10.1210\/mend.13.11.0367","type":"journal-article","created":{"date-parts":[[2014,1,8]],"date-time":"2014-01-08T17:29:56Z","timestamp":1389202196000},"page":"1864-1872","source":"Crossref","is-referenced-by-count":6,"title":["Characterization of the Thyroxine-Binding Site of Thyroxine-Binding Globulin by Site-Directed Mutagenesis"],"prefix":"10.1210","volume":"13","author":[{"given":"Christoph","family":"Buettner","sequence":"first","affiliation":[{"name":"Department of Medicine Klinikum Innenstadt Ludwig-Maximilians-University D-80336 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Helmut","family":"Grasberger","sequence":"additional","affiliation":[{"name":"Department of Medicine Klinikum Innenstadt Ludwig-Maximilians-University D-80336 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kristine","family":"Hermansdorfer","sequence":"additional","affiliation":[{"name":"Department of Medicine Klinikum Innenstadt Ludwig-Maximilians-University D-80336 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bingkun","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Medicine Klinikum Innenstadt Ludwig-Maximilians-University D-80336 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bettina","family":"Treske","sequence":"additional","affiliation":[{"name":"Department of Medicine Klinikum Innenstadt Ludwig-Maximilians-University D-80336 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Onno E.","family":"Janssen","sequence":"additional","affiliation":[{"name":"Department of Medicine Klinikum Innenstadt Ludwig-Maximilians-University D-80336 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"80","reference":[{"key":"2020071615005699300_R1","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1056\/NEJM196805232782107","article-title":"Role of plasma proteins in the binding, distribution, and metabolism of the thyroid hormones.","volume":"278","author":"Oppenheimer","year":"1968","journal-title":"N Engl J Med"},{"key":"2020071615005699300_R2","first-page":"541","article-title":"Transport, cellular uptake, metabolism of thyroid hormone","author":"Refetoff","year":"1989"},{"key":"2020071615005699300_R3","doi-asserted-by":"crossref","first-page":"3558","DOI":"10.1016\/S0021-9258(17)33380-X","article-title":"Thyroxine-protein interactions. 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