A conserved acidic residue drives thyroxine synthesis within thyroglobulin and other protein precursors

Abstract

Thyroxine, the main hormone product of the thyroid, is produced at multiple sites within its protein precursor thyroglobulin. Each site consists of two tyrosine residues which undergo iodination and coupling, resulting in the synthesis of thyroxine at the acceptor tyrosine, where the hormone synthesis is later completed by proteolysis. Within the structurally resolved sites, the role of an essential conserved acidic residue preceding the acceptor remains elusive. To elucidate the mechanism of thyroxine synthesis we engineered a single-site minimal protein precursor. First, by its in vitro iodination and site-directed mutagenesis we show that the presence of the acidic residue, preferably glutamate, favors thyroxine synthesis. Secondly, within the designed precursor, we computationally modeled the reaction of iodination and iodotyrosine coupling giving rise to thyroxine. Our results reveal that hormone formation is triggered by iodotyrosine deprotonation, facilitated by proximity to a carboxylic group, closer in the case of glutamate, in line with our experimental findings and sequence conservation. Hereafter, we surmise that in the natural precursor thyroglobulin, two evolutionary late and slower hormonogenic sites coexist with an early evolutionary and faster one. Indeed, the latter is overlapping with a proteolytic site, thereby allowing prompt thyroxine release from thyroglobulin.

Keywords: density functional theory; iodination; molecular dynamics; protein engineering; thyroglobulin; thyroid hormones; thyroxine.

Figure 1

In vitro T4 synthesis on a model precursor.A, sequence conservation of hormonogenic sites (A), (B), and (D) across vertebrate TG. The consensus shows that at site A the acceptor tyrosine is always preceded by a glutamate, while in the evolutionary late sites (B) and (D) the consensus sequence favors an aspartate. The lower panel indicates the mutants of the MBP precursor that have been engineered to investigate the impact of the residue preceding the acceptor on T4 synthesis. B, schematics showing the MBP model precursor (yellow) and its engineered hormonogenic tyrosine pair (Y341 donor in gray and acceptor Y375 in teal), together with the general overview of T4 synthesis steps. C and D, dot blot, and T4-ELISA analysis of iodinated MBP variants indicate that E/D facilitate T4 synthesis over K/S at residue 374. Measurements were performed in three biological replicates.

Figure 2

Molecular Dynamics simulations of T4 synthesis within model precursor variants.A, Molecular Dynamics simulations of the model T4 precursor MBP, showing starting and representative frames of iodinated, distally iodinated (DIT from the start of the simulation) and proximally iodinated (iodinated at the most probable conformation) hormonogenic tyrosine pairs. B, probability distributions tracking the tyrosine-tyrosine distance O_ζ-C_β representative of the coupling process, for every MBP variant (where the residue preceding the acceptor 374 is a E, D, K, S) and in the absence of presence of presence of the iodine moiety. The time-dependent graphs are reported in Figs. S5–S7.

Figure 3

Mechanism of T4 synthesis derived by in silico simulations of the MBP model precursor.A, calculated mechanisms for DIT coupling following a sequential (ΔG1) or concerted (ΔG2) route. Both mechanisms are triggered by a preliminary parallel to-perpendicular stacking rearrangement of the DITs couple (see Fig. S9 for details). B, ΔG1 values calculated for the MBP variant E374 considering a radical, anionic, and hybrid process coming from the donor (Y341) or as the real system from the acceptor (Y375). Radical processes are way more favored over anionic ones (see Figs. S8 for further details). C, ΔG2 values were calculated for all MBP mutants, showing that, in line with experimental data, the reaction is facilitated by the presence of a E, D, K, S preceding the acceptor in this order. D, estimated pKa for MIT and DIT, taking phenol as reference (see Fig. S12 for details). E, the probability distribution of distances (expressed in Angstrom) between the hydroxyl group oxygen atom of the acceptor tyrosine 375 (O_ζ) and the carbon atom belonging to the carboxylic moiety of the sidechain of the acidic residue 374 (i.e.., C_δ of glutamate in red, C_γ of aspartate in green). The distributions have been computed for the three configurations and iodination conditions inspected in the present study and described in the Experimental Procedures section, i.e., from left to right, distal non-iodinated tyrosines, distal iodinated tyrosines and proximal iodinated tyrosines.

Figure 4

Role of the consensus sequence Glu/Asp-Tyr in T4 formation within protein precursors.A, our model describes the role of an acidic residue (mainly glutamate) preceding the acceptor in facilitating deprotonation and driving directional T4 synthesis. B, our rationale for the coexistence of multiple T4 sites in TG: site A is flanked by an exposed cathepsin residue (early synthesis and release in the extracellular lumen of thyroid follicles), site B, D, and A (release in the lysosomes after endocytosis).