Structural Mechanism Of Hormone Binding And Release From Thyroxine-binding Globulin

Thyroxine (T4) binding-globulin (TBG) has a tight binding affinity for T4 (Kd～0.1 nM). In native state, it binds 75% of serum T4 and T3 and serves as an extrathyroidal reservoir of thyroid hormone. TBG can actively release the thyroid hormone in localized region by the cleavage of the reactive centre loop (RCL). The binding and release of hormones from TBG is regulated by an allosteric mechanism based on the configuration between A-sheet and the reactive loop.To investigate how the changes in RCL are transmitted to the hormone binding site we developed a sensitive fluorescent method for measuring binding affinity of TBG. L-thyroxine-6-carboxyfluoroscein (termed T4-6-CF) was synthesized, which has the lower affinity, but the the same orientation and contacts within the thyroxine-binding pocket as unmodified thyroxine. When the conformation of TBG is altered, T4-6-CF has the similar Kd change times as T4 (5.5 vs 5.0). The binding characteristics of T4-6-CF with TBG can represent that of T4 with TBG.The transmitting process of the allosteric mechanism is investigated by crystallization and site-mutagenesis. Shortening of the connecting loop from helix D to strand 2 of theβ-sheet A (hD-s2A) leads to an increased Kd (24.9nM vs normal 2.2nM) and a decreased Kd change time after cleaved (2 times vs normal 5.5 times). The replacement of key residues, Lys243 in the loop connecting strand 2 and 3 ofβ- sheet B (s2/3B loop) and Arg378 in the loop connecting strand 4 and 5 ofβ-sheet B (s4/5B loop), results in the similar Kd change. Overall, the RCL-P-sheet A configuration influences hD-s2A loop. This loop is linked to residues in the s2/3B loop notably including Lys243, and with the s2/3B loop in turn interacting with the s4/5B loop, especially so with Arg378, that directly flanks the binding site. It is the close packing of these loops that allows the transmission of the expansion of theβ-sheet A through the hD-s2A loop, s2/3B loop and Arg378 to give a destabilisation of the binding pocket that in TBG will favour the release of the tightly-fitting thyroxine.Another transmitting pathway may be regulated by cation-pi interaction between Arg381 and concerned aromatic rings. The Arg381 is replaced with serial amino acids. The Lys381 mutant increases the binding affinity (0.25nM vs normal 1.69nM) and other mutants such as Gln381 decrease the binding affinity (212.45nM vs normal 1.69nM). The critical residue Tyr20 is substituted by Ala, which results in an almost unchanged Kd (1.67nM vs normal 1.69nM) in native state and decreased Kd change time (6.13 times vs normal 14.45 times) in cleaved state. The superposition and the comparison of accessible surface area (ASA) between TBG and cleaved TBG show that the twisting occurs in Helix A after cleaved. Overall, in native unliganded state, Tyr20 is too far from the binding pocket to form cation-pi interaction with Arg381, and the flexible Arg381 hovers in the pocket and prevents non-aromatic ligands from binding. Upon aromatic ligand approaching, cation-pi interaction is formed, Arg381 is reoriented and the binding pocket is opened. Then the ligand is fitted to the pocket and the cation-pi interaction is retained to strengthen the binding. Once TBG is cleaved in reactive centre loop, twisting occurs in helix A. Tyr20 is reoriented and become enough close to binding pocket to form cation-pi interaction with Arg381. The competition of Tyr20 for Arg381 to form cation-pi interaction impairs the cation-pi interaction between Arg381 and ligand and decreases the binding affinity of TBG for iigand. This results in the ligand release. The twisting of Helix A and the insertion of reactive centre loop intoβ-sheet A may be linked by the ionic bonds between Lys302 and Glu40 which are conserved completely in all listed species.We investigate how the temperature change affects the thyroid hormone release from TBG. The Kd with TBG is measured at 22℃,37℃,39℃and 42℃, respectively. With the temperature increase, the Kd increases gradually. The Kd at 37℃is set to be control. The sensitivity of Kd to temperature is expressed by KdT/Kd37-Obviously, Kd is more sensitive to temperature in range from 37℃to 39℃compared to other ranges. TBG mutant, Ala191 replaced with Thr, in the aborigines who live in a drought and hot land in Australia has evolved TBG to adapt to the hot climate. The high frequency TBG-Ala191Thr variant in aborigines has lower temperature sensitivity than TBG. Overall, TBG is involved in the regulation of free T4 during body temperature change. This delicate regulation is a complement for classical hypothalamic/pituitary/thyroid axis. TBG variant in extreme climate proves that free T4 regulation by TBG is important for body temperature equilibrium in new set point. This free T4 regulation by TBG is based on the protein motion. The flip-flop change of loop and the twisting of TBG regulate the binding of thyroxine. Temperature change affects the two movement and then results in the binding and release of thyroxine.In summary, we investigate the mechanism of thyroxine binding and release for cleaved TBG and native TBG. The understanding of the cleaved release in TBG benefits our design about drug-targeted delivery system based on TBG. The new knowledge about the thyroxine release in native TBG tells us that TBG is involved in the regulation of free T4 during body temperature change, which is a complement for classical hypothalamic/pituitary/thyroid axis.