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, 103 (36), 13321-6

Structural Mechanism for the Carriage and Release of Thyroxine in the Blood

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Structural Mechanism for the Carriage and Release of Thyroxine in the Blood

Aiwu Zhou et al. Proc Natl Acad Sci U S A.

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 A, 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 beta-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.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
TBG and the thyroxine binding site. (a) Structure of TBG with thyroxine (space-filled). The upper half of the Aβ-sheet (blue) is opened, with initial insertion of the reactive loop (red) to P14 threonine, 14 residues before the reactive center P1. (b) Binding pocket showing thyroxine in stick form enclosed between strands 3–5 of the B-sheet and helices H and A and with iodine atoms, contoured at 5 times rms density in a log-likelihood gradient map for anomalous scattering (16).
Fig. 2.
Fig. 2.
Binding and triggered release of thyroxine. (a) Interactions with adjacent side chains anchor thyroxine within the pocket. Thyroxine release will be triggered on full insertion of P14 threonine (space-filled upper left) displacing Tyr-241 and disrupting the H-bonds that anchor thyroxine and the flanking peptide loop between s4B and s5B (blue). This network will be similarly disrupted by the common presence (19) in Australian aborigines of a Thr at 191 (circled red; see also Fig. 4). (b) The triggered movement of the flanking s4B–5B loop is shown in the homologous pocket in antichymotrypsin. The open pocket of TBG before loop insertion (blue) matches that of active antichymotrypsin (gray). Transition to the fully inserted loop (cyan) or to the partially inserted δ-form (green) in antichymotrypsin both result in a 4- to 5-Å shift of the loop with a contraction of the binding pocket.
Fig. 3.
Fig. 3.
Stereoview of thyroxine-binding pocket showing hydrophobic and H-bonding interactions. The replacement of Leu-246 by Thr in the recombinant variant mD–TBG results in a marked reduction in binding affinity (14) with the replacement being compounded by a new glycosylation site at Asn-244, a residue that stabilizes the peptide loop flanking the binding site (see also Figs. 2a and 4a).
Fig. 4.
Fig. 4.
Thyroxine binding and mutations. (a) Space-filling depictions show how the side chain of Arg-381 stacks with the outer phenolic ring of the thyroxine and how the mutation of Ser-23 → Thr will sterically hinder the binding of thyroxine. Mutations in CBG causing a loss of hormone binding (at Trp-371 and Asp-367 in CBG) affect the structural equivalents of Arg-381 and Glu-377 in the TBG pocket. (b) The mutation Ala-191 → Thr is commonly present in West Australian aboriginals (TBG inheritance is X-linked: 56% of men were hemizygotes for the variant; 29% of women were homozygotes and 38% heterozygotes; ref. 19). As modeled here, Thr-191 will predictably compete for the H-bond network formed with Arg-378 (circled in a) that anchors thyroxine. A concomitant mutation in the aboriginal, Leu-283 → Phe, in an adjacent tightly packed region of the molecule, will exacerbate the perturbation of the binding site.
Fig. 5.
Fig. 5.
Reversibility of the conformational transition in TBG. (a) The A-sheet in TBG is blocked at the level of entry of the P8 residue of the reactive loop by a barrier centered on His-331 that cannot be readily displaced by the P8 proline uniquely present in TBG. (b) In other serpins, including antichymotrypsin (brown), the sheet is only partly opened (circled). (c) Entry of the loop to P12 in δ-antichymotrypsin (brown) requires a complete opening of its sheet. However, in TBG (blue), the sheet is already opened to allow ready movement of the loop to P10 without disruption of the His-331 barrier. (d) Schematic of flip-flop conformational equilibrium, with unbound TBG modeled on δ-antichymotrypsin on the left and the structure of thyroxine-bound TBG solved here on the right. (e) Transverse urea gradient gels confirm δ-equivalence of unbound TBG. (i) Typical serpin profile (α-1-antitrypsin), with unfolding near 1 M urea. (ii) TBG (without thyroxine) has a δ-conformation stabilized profile with unfolding at 3 M urea (22). (iii) Identical to that of the low-affinity TBG+3 mutant (25). The longer loop in the TBG+3 mutant also allows conversion in the urea gel with complete insertion of the loop to give the hyperstable latent form seen in iii as a second unbroken profile.

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