The TSH receptor (TSHR) on the surface of thyrocytes is an important regulator of thyroid growth, development, hormone synthesis, and secretion. It is also the primary target of autoantibodies in Graves’ disease (autoimmune hyperthyroidism; Davies et al., 2020). From cloning, sequence analysis, partial crystallization, and biochemical studies, this GPCR has been deduced to be made of a large ectodomain (ECD) and membrane-bound signal transducing transmembrane domain (TMD; Rapoport et al., 1998; Davies et al., 2005). The ECD is further divided into a leucine-rich domain (LRD) forming a curved structure which is linked to the TMD by a 130 amino acid (AA) linker region (LR) known commonly as the ‘hinge region’” (AA280-410). Unique to the TSHR is a large 50 AA cleavage region (AA316-366) located within the LR that is proteolytically degraded leaving a cleaved ECD thought to be tethered to the TMD via three cysteine bonds (Tanaka et al., 1999; Tanaka et al., 2001) and sometimes referred to as the C peptide.

Stimulating TSHR antibodies only recognize the TSHR in its native form (Latif et al., 2012) indicating that the receptor-epitope must be in a conformation that the antibody can recognize. Crystallization studies (Sanders et al., 2007; Sanders et al., 2011), besides producing crystal structures for the LRD, have also shown that antibodies, which either stimulate or block TSHR signaling, only bind to the LRD when the receptor is conformationally correct and can compete for TSH binding. In contrast, ‘neutral’ antibodies to the TSHR which do not initiate a traditional signal (Morshed et al., 2018) nor inhibit TSH binding, predominantly, but not exclusively, bind to linear epitopes in the LR (Sun, 2018). Although the partial LRD structure has been determined with x-ray crystallography (Miller-Gallacher et al., 2019), no experimental structure has been found for the LR, and until recently, only partial models have been proposed (Kleinau et al., 2013; Kleinau et al., 2017; Morshed et al., 2009). On the basis of the immune response to the TSHR we, and others, have suggested that the LR is not an inert scaffold but rather an important ligand-specific structural and functional entity (Schaarschmidt et al., 2014), but its structure has not been examined in the context of the full-length receptor. However, the recent success of the artificial intelligence (AI)-based Alphafold2 (Jumper et al., 2021) program led us to believe that it might be possible to generate a full-length receptor structure by combining the LRD-LR structure generated by Alphafold2 (that includes a structural model of the LR region but no TMD and for which neither experimental nor homology models are available) with our recently published model of the TSHR TMD (named TRIO; Mezei et al., 2021). Note, that Alphafold2 provided a separate model for the TMD and did not provide a fully assembled structure. Therefore, we decided to use our own model as it was already equilibrated in the membrane environment and partially based on experimental validation. This full-length model could then be enhanced and verified with molecular dynamics (MDs) simulation. We can now report a successful computer-based approach to obtain insight into the LR allowing us to complete a full-length model of the TSHR. We have examined the behavior of this TSHR in a lipid-embedded, electro-neutral, aqueous environment by MD simulation studies and showed that the LR is indeed an intrinsically disordered protein but can be stabilized by TSH ligand binding to the LRD.

The TSHR, similar to the FSH and LH/hCG receptors, consists of LRD, a large extracellular ligand binding domain incorporating 11 repeats, and a TMD, linked via a 130 AA LR. The TMD is made up of eight helices joined by extracellular and intracellular loops and a long C-terminal cytoplasmic tail. The TMD is embedded in a phospholipid bilayer and transduces a cascade of signals by engaging several different G proteins (Laugwitz et al., 1996) and β arrestins (Boutin et al., 2014; Frenzel et al., 2006). The interest in the TSHR has been largely fueled by its role as a major human autoantigen in autoimmune thyroid disease, especially Graves’ disease (Davies et al., 2020).

Detailed mapping of binding sites and interaction partners for the TSHR ligand, TSH, and for stimulating and blocking monoclonal autoantibodies to the TSHR, have been revealed by homology modeling (Núñez Miguel et al., 2004) and crystallization of the partial ECD bound to these autoantibodies (Sanders et al., 2007; Sanders et al., 2011). Furthermore, homology modeling has suggested possible mechanisms by which activation of the receptor by TSH and a stimulating antibody might occur (Jiang et al., 2014; Kleinau and Krause, 2009; Krause et al., 2012). However, all these tripartite models have remained incomplete due to the lack of a reasonable structure for the large TSHR LR/hinge region. Here, we present a full-length model of the TSHR which became possible because of the recent availability of models of the human proteome generated by the AI-based protein folding program Alphafold2 (Jumper et al., 2021). We combined the Aplhafold2-generated structure of the LRD-LR complex with our recent MD-refined homology model of the TSHR TMD (Mezei et al., 2021) and further refined the structure with MDs in a DPCC membrane environment. Given that the structures of the LRD and the TMD of the TSHR have been described in earlier studies (Ali et al., 2015; Latif et al., 2015), our analysis in this report was firstly focused on the structure of the LR, its intramolecular and molecular bonding dynamics, and its structural variations in lieu with LRD and TMD structures.

It is notable that in the initial structure obtained with our heuristic process by the combination of the two parts using the relative orientation of the LRD (residues 22–280) and the TMD (residues 409–694) resulted in a full-length receptor model that retained the relative orientation of the ECD and the TMD in spite of the large conformational fluctuation of the LR (Figure 5) and the large fluctuation on the angle between the LR and the LRD (Figure 8) supporting the overall correctness of our initial model. Further support can be seen in the fact that the relative orientation of the ECD and the TMD allows the formation of TMD dimers in a conformation predicted and experimentally verified by our earlier work (Latif et al., 2015) and which would still leave the LRD-binding surface free for ligand or autoantibody binding. However, in the Alphafold2-predicted conformation presented here, the concave surface of the LRD where autoantibodies bind, is partly occluded by the LR and thus would clash with a bound TSHR antibody whose binding sites on the LRD are known (PDB id 3g04). This conundrum was resolved by the observations that (a) the structure of the LR cluster is highly flexible and (b) the relative orientation of the LRD with the rest of the structure is also highly variable resulting in a significant population of conformations where stimulating and blocking TSHR autoantibodies could access the receptor to activate or block signaling. Thus, we can say that the Alphafold2 structure of the LR, while useful in providing a starting point for the MD simulation, was not correct in the sense that it missed the large conformational freedom of the LR needed for ligand binding. Given the low reliability score assigned to the LR part of the Alphafold2 structure, this observation was not a surprise. We also submitted the LR sequence to the PONDR (Predictors of Natural Disordered Regions) server (http://www.pondr.com/) – using the methods VLXT (the default), VL3 NNP, and VL2 NNP, and it predicted that 28.91%, 64.81, and 62.5%, respectively, of the LR is disordered. Furthermore, these conclusions could be further verified by obtaining, if possible, cryo electron microscopy (CEM) or a crystal structure of the native full-length TSHR perhaps stabilized by an autoantibody to the LR.

Based on this study, we can conclude that the LR in the TSHR is remarkably flexible, sampling vastly different overall conformations without settling on a well-defined tertiary structure. While different SSEs formed during the simulation (mostly helices), they were transient, as seen from Figure 6A. On the other hand, contacts between the LR and the LRD persisted throughout the simulation. The conclusion is that the LR is an IDP. This conclusion is consistent both with the fact that so far no one has succeeded in obtaining its crystal structure and with the suggestion (Jumper et al., 2021) that low reliability scores indicate that the protein is intrinsically disordered. However, from these studies, we speculate that the highly flexible nature of the LR is what allows it to accommodate both the ligand and the autoantibodies to the LRD.

The fact that the two pairs of cysteines stayed close with their putative partners for disulfide bonding indicated that these cysteines should form a disulfide bond. Furthermore, the fact that when the TSH was added to this model, these cysteines moved apart also suggested that they could be part of the signal-transduction mechanism.

The convergence of the simulation is always an open question. However, there are important indicators that suggest that our sampling was adequate. Figure 7, the history of hydrogen bonds involving the LR, showed that most of the bonds had formed during the first half of the simulation; no new ones formed in the last quarter. Hydrogen bonds also kept forming and reforming. Similarly, Figure 6A shows that most SSEs formed and broke several times during the simulation. Taken together, these indicators allow us to conclude that the simulation involved adequate sampling.

In addition to our examination of the LR structure, we compared the changes seen in the TMD helix bundle, from the respective reference structure in the TMD-only simulation, versus the full-length model. It was of great interest that the change in the curvature (and, as a consequence, in the end-to-end distance) of helix 3 was significantly greater in the new full-length model than in the TMD-only model. Comparing the range of values sampled in the representative structures (data not shown), we found similar differences. This observation supported the hypothesis put forward earlier (Davies and Latif, 2015) that helix 3 is highly important for the signal transduction of the TSHR and consistent with a variety of small molecule activators which all interact with helix 3 (not illustrated). We also examined the LR cysteines. Much has been discussed concerning the role of cysteine bonds in anchoring the LR to the LRD following post-receptor processing which involves cleavage of the unique 55 AA insert in the LR (see Figure 9C). The analysis of the cysteines in the LR showed the remarkable affinity of one pair to each other (C283-C398) and to a lesser degree for the second pair (C384-C408). However, the third pair (C301-C390) showed poor affinity, leading to the suggestion that in the fully formed TSHR, only two of the cysteine bonds were formed.

Recently, structures of the luteinizing hormone receptor (LHR) in complex with different proteins and in its inactive state, a total of five structures, obtained by CEM have been described (Duan et al., 2021). In accordance with our simulation, the relative orientation of the LRD with the TMD was quite variable. The LR in the LHR, however, while significantly shorter than the LR in the TSHR, was missing more than 40 residues in the CEM structures, thus preventing detailed comparison with the TSHR LR. It is notable that the short helix at the N-terminal end of the LR was present in all five CEM structures and during the 1000 ns MD run described here. Also, the LR conformations in the CEM structures were very different from each other, reflecting the conformational variations observed during our TSHR MD run. Interestingly, our simulation of 1000 ns with the heterodimeric TSH showed stabilization of SSEs, which was clearly absent without TSH (Figure 6), further suggesting that the LR plays an important part in TSH action and that TSH is able to stabilize the flexible LR region after transitioning through various dynamic structural changes. Furthermore, the separation of the LR and the TMD during the simulation suggests that for the proper action of the TSHR on binding of the TSH, then at least one cysteine bond has to be formed.

Our simulation of the full-length TSHR embedded in a lipid membrane, solvated with water-containing counterions, i.e., in a biologically relevant environment suggested (a) the LRD and the TMD continue to maintain their fold; (b) the LR is flexible, but maintains protein-like behavior forming secondary structure elements that are, however, transient; and (c) the relative orientation of the LRD is also variable. Both the orientation of the LRD and the structural flexibility of the LR suggests that these features are likely to be important for the TSHR to accommodate the diverse ligands such as TSH and autoantibodies that are known to bind to the extracellular region of TSHR.

Our preliminary results from a simulation of the TSHR complexed with its ligand TSH showed that TSH formed strong interactions with both the LRD and the LR. Furthermore, it appeared to increase the longevity of the secondary structure elements in the LR. In addition, the breakup of the LR-TMD interface points to the importance of the disulfide bonds in the LR.

In conclusion, we performed the first MDs simulation of the full-length TSHR that includes the characterization of a flexible LR and concluded that the LR is constitutively unstable in the native state of and receptor thus can be considered an IDP. The earlier generated models (e.g. Núñez Miguel et al., 2004) only represented one conformation of the many LR structures as well as missing the 50 AA insert unique to the TSHR and also highlights the importance of considering conformational ensembles as the most accurate model of the LR. In addition, our preliminary results indicate that TSH is able to stabilize this disordered protein, which suggests that stabilization of the LR is important for signaling to ensue.

The initial model generated is available from the Dryad server; URL: https://doi.org/10.5061/dryad.rjdfn2zdp. The software used for the analysis are available at the URL https://mezeim01.u.hpc.mssm.edu.

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Author details

1. Mihaly Mezei

   1. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, United States
   2. Department of Medicine, Thyroid Research Unit, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   Conceptualization, Software, Formal analysis, Methodology, Writing – original draft, Writing – review and editing

   For correspondence

   Mihaly.Mezei@mssm.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0294-4307

2. Rauf Latif

   1. Department of Medicine, Thyroid Research Unit, Icahn School of Medicine at Mount Sinai, New York, United States
   2. James J. Peters VA Medical Center, New York, United States

   Contribution

   Conceptualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4226-3728

3. Terry F Davies

   1. Department of Medicine, Thyroid Research Unit, Icahn School of Medicine at Mount Sinai, New York, United States
   2. James J. Peters VA Medical Center, New York, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Writing – review and editing

   For correspondence

   terry.davies@mssm.edu

   Competing interests

   TFD is member of the Board of Kronus Inc (Starr, ID, USA)

   "This ORCID iD identifies the author of this article:" 0000-0003-3909-2750

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