Browsing by Author "Sauer-Eriksson, A. Elisabeth"
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- High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxinePublication . Eneqvist, Therese; Lundberg, Erik; Karlsson, Anders; Huang, Shenghua; Santos, Cecilia; Power, Deborah; Sauer-Eriksson, A. ElisabethTransthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3 -triiodo- L-thyronine (T3) and 3,5,3 ,5 -tetraiodo-L-thyronine (thyroxine, T4). Human TTR has higher affinity for T4 than T3, whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 Å resolution and bound to ligands T3 and T4, both at 1.9 Å resolution. The apo structure is similar to human TTR with structural changes only at -strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR T4 complex shows the T4-binding site to be similar but not identical to human TTR, whereas the TTR T3 complex shows the I3 halogen situated at the site normally occupied by the hydroxyl group of T4. The significantly wider entrance of the hormone- binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T3 and T4.
- Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminalPublication . Morgado, Isabel; Melo, Eduardo P.; Lundberg, Erik; Estrela, Nídia L.; Sauer-Eriksson, A. Elisabeth; Power, DeborahTransthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild-type sea bream TTR (sbTTRWT) plus two mutants in which six (sbTTRM6) and twelve (sbTTRM12) N-terminal residues were removed. Ligandbinding studies revealed similar affinities for T3 (Kd=10.6±1.7nM) and T4 (Kd=9.8±0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3±15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine-T.In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.
- Thyroid hormone binding by recombinant sea bream transthyretin: the role of the N-terminal regionPublication . Morgado, Isabel; Sauer-Eriksson, A. Elisabeth; Power, DeborahTransthyretin (TTR) along with thyroxine-binding globulin (TBG) and albumin (ALB) constitute Thyroid Hormone-Binding Proteins (THBP) in vertebrates. These proteins bind and transport thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood. THBP are poorly characterized in fish and in the present study binding of THs by sea bream TTR was determined. Teleost TTR is a single polypeptide chain of 130 amino acids, but in common with mammals the functional form in the plasma is a tetramer. In contrast to mammalian TTR (127 amino acids), fish TTR has a longer N-terminal region and the latter has been proposed to influence TH binding. In the present study recombinant sea bream TTR (sbTTR, wild type), plus two recombinant N-terminal mutants were produced and purified. Binding of [I125]-T3 to the purified TTRs was confirmed by polyacrylamide gel electrophoresis under nondenaturing conditions followed by autoradiography and confirmed all bind THs as a tetramer. Ligand binding studies with labeled [I125]-T3 were performed and revealed that [I125]-T3 was displaced in a similar way from wild type and mutant TTRs by increasing concentrations of unlabeled T3. Similar Kd values were obtained for both T3 and T4 binding to wild type and mutant TTRs indicating that the N-terminal region does not seem to be important for the binding characteristics of sbTTR.