Structural mechanism for the carriage and release of thyroxine in the blood

Abstract

The hormones that most directly control tissue activities in health and disease are delivered by two noninhibitory members of the serpin family of protease inhibitors, thyroxine-binding globulin (TBG) and corticosteroid-binding globulin. The structure of TBG bound to tetra-iodo thyroxine, solved here at 2.8 Å, shows how the thyroxine is carried in a surface pocket on the molecule. This unexpected binding site is confirmed by mutations associated with a loss of hormone binding in both TBG and also homologously in corticosteroid-binding globulin. TBG strikingly differs from other serpins in having the upper half of its main ␤-sheet fully opened, so its reactive center peptide loop can readily move in and out of the sheet to give an equilibrated binding and release of thyroxine. The entry of the loop triggers a conformational change, with a linked contraction of the binding pocket and release of the bound thyroxine. The ready reversibility of this change is due to the unique presence in the reactive loop of TBG of a proline that impedes the full and irreversible entry of the loop that occurs in other serpins. Thus, TBG has adapted the serpin inhibitory mechanism to give a reversible flip-flop transition, from a high-affinity to a low-affinity form. The complexity and ready triggering of this conformational mechanism strongly indicates that TBG has evolved to allow a modulated and targeted delivery of thyroxine to the tissues.corticosteroid-binding globulin ͉ serpin ͉ thyroxine-binding globulin ͉ crystal structure T hyroxine is the major hormone controlling cellular development as well as the rate of body metabolism. It is a small molecule formed by the linkage of two tyrosines, which are iodinated to give the alternative tri-or tetra-iodo forms of the hormone. Thyroxine is principally carried in the blood by thyroxine-binding globulin (TBG), which binds it with high affinity (K d ϭ 0.1 nM) in equilibrium with steady-state low levels of free thyroxine (1-3). Although TBG is not a protease inhibitor, it is otherwise a typical member of the serpin family of protease inhibitors. The alignment of its sequence shows that it retains the same framework structure as the archetypal inhibitory members of the family, ␣ 1 -antitrypsin, antithrombin, and antichymotrypsin (4). It has similarly retained a typical reactive site loop, with a putative reactive center at the position denoted P1 and, with 17 residues before it, the hinge of the loop at P17 (see Fig. 1 and Table 1). In particular, TBG undergoes the profound and irreversible conformational change on cleavage of its reactive loop, which is characteristic of the serpins (5, 6). Such cleavage of the reactive loop of TBG by proteases does occur during sepsis to give a 3-fold reduction in its binding affinity (7-9). However, because only a minor proportion, Ͻ20%, of the circulating TBG is bound to thyroxine, even this relatively small decrease in affinity will result in an effective release of thyroxine (10), as demonstrably occurs at sites of inf lammation (11). Normally, ho...

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

“…In most serpins, P8 is represented by a threonine with a small side chain (6). From the crystal structure of thyroxin binding globulin, it has been suggested that the proline at P8 limits loop insertion within the upper half of the ␤-sheet A and that in most serpins the barrier formed by a highly conserved histidine, in the lower halves of strand s3A and s5A, is broken by the side chain of a P8 threonine upon entry of the loop (34). Although there is no direct evidence for this, our results support it because substituting the P8 threonine in human CBG with proline appears to result in limited loop insertion upon cleavage.…”

Binding interactions of perfluoroalkyl substances with thyroid hormone transport proteins and potential toxicological implications

“…The binding data were used to determine K d values of 8 nM and 27 nM for the two binding sites of TTR (R 2 = 0.98), and K d values of 498 and 524 nM were obtained between F-T4 and TTRmutK15G for the two binding sites (R 2 = 0.99). For TBG, there is only one T4 binding site (Zhou et al, 2006). Similarly, the fluorescence polarization enhancement was 190, 260, and 250 mP, as the concentration of TBG, TBGmutR378G, or TBGmutR381G increased to 1 mM, respectively (Fig.…”

“…OH-PBDEs with medium size had the highest binding affinity to TTR, whereas the OH-PBDEs with the larger size are optimal for TBG binding (Cao et al, 2010;Ren and Guo, 2012). The different binding patterns of ligands to TTR and TBG might be due to their different patterns of T4 binding pocket, which is a hydrophobic channel in TTR but a surface pocket in TBG (Hamilton et al, 1993;Zhou et al, 2006). According to several TBG-ligands crystal results, both residues R378 and R381 of TBG play crucial roles in the interaction with ligands (Zhou et al, 2006;Zheng, in press).…”

“…The different binding patterns of ligands to TTR and TBG might be due to their different patterns of T4 binding pocket, which is a hydrophobic channel in TTR but a surface pocket in TBG (Hamilton et al, 1993;Zhou et al, 2006). According to several TBG-ligands crystal results, both residues R378 and R381 of TBG play crucial roles in the interaction with ligands (Zhou et al, 2006;Zheng, in press). Here, we found mutation of R378 or R381 to glycine resulted in significantly weaker binding of these two TBG mutants towards PFTA and PFTdA when compared with the binding affinities of the wild-type protein (Table 2).…”

Crystallization and crystallographic studies of kallistatin

“…As serpins are known to adopt different conformations, models of protein C inhibitor Li et al, 2007) and thyroxine-binding globulin (Zhou et al, 2006), representing one relaxed and two stressed serpin conformations ($47% sequence identity; PDB entries 1lq8, 2ce0 and 2hi9), were selected as search models. A clear single solution was obtained from a search using PDB entry 1lq8 as the model with one copy of molecule in the asymmetric unit, indicating a solvent content of $60%.…”