Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation

Abstract

The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor.

Keywords: G protein-coupled receptor (GPCR); cell signaling; computer modeling; mass spectrometry (MS); protein cross-linking; receptor structure-function; site-directed mutagenesis; structural biology; surface plasmon resonance (SPR).

FIGURE 1.

Schematic representation and identified cross-links of the TSHR·TSH complex. A, schematic representation of the TSHR·TSH complex including disulfides, cross-linked residues identified by mass spectrometry and significant residues of the TSHR, including residues with reported constitutively activating mutations (Ser-281, Ile-486, Ile-568), the sulfation site (Tyr-385), and boundaries of the model within the HR (Phe-381, Ser-304). The respective spacer-length of the cross-linking reagents is specified in the figure legend. B, boxplot of Cβ distance distribution between residues connected by chemical cross-linking within the homology models of the TSHR-ECD·TSH complex. The employed cross-link-specific cutoff distance (Table 1) is indicated by a dashed line in gray. Cross-links 8 and 9 include one residue located in the part of the HR not included within the models. For these, the distance to the closest residue included in the models is reported, and the missing residues are considered in the cutoff distance. C, cross-links (green lines) between the hinge region of the TSHR (blue) and the hormone (red, α-chain; yellow, β-chain) suggest that the HR, including the part not resolved in the FSHR-ECD/FSH template and, therefore, not included in the homology models, is oriented toward the hormone and most likely also contributes to ligand binding.

FIGURE 2.

Strategy for generating full-length GPHR models. An integrated structural biology approach combining computational techniques (A–D) with experimental data (E1 and E2). Homology models were constructed using Rosetta 3 for the TSHR-ECD (A1 and A2) and the multitemplate approach of RosettaCM for the TSHR-TMD (B1). Chemical cross-linking of the soluble TSHR-ECD yielded 17 cross-links that were used to guide template selection and evaluate the models of the TSHR-ECD (E1). The model sets were further analyzed by clustering analysis using Calibur (A3 and B2). Models were selected based on energy and cluster size. The combination of 30 TSHR-TMD models with 5 TSHR-ECD models by docking yielded 150,000 docked models (C1). During docking a cross-interface disulfide between Cys-284 and Cys-408 was enforced. From the docked poses ∼100 models were selected based on interface score and agreement with the experimentally verified contact of Ser-281 with Ile-486 (E2) for reconstruction of the linker region (Lys-401–Ile-411, C2). The model set of the full-length TSHR was further analyzed by contact maps (D1) and clustering (D2). Feasibility of the full-length models was verified by reintroduction of the ligand and remodeling of the thumb region (D2).

FIGURE 3.

Homology models of the TSHR-ECD (blue)/TSH (red, α-chain; yellow, β-chain) complex. Cross-links (green dotted lines) confirm the fold (A) of the domains and (B) a similar binding mode of TSH as observed for FSH to the FSHR-ECD. C, the cross-link (green line) between the N terminus of the TSH α-chain and Lys-101 close to the C terminus of the β-chain suggests close proximity of the termini. D, cross-link to Lys-45 of the TSH α-chain are not met by any homology model of the TSHR-ECD·TSH complex. E, homology models suggest a potential TSHR-specific contact between Glu-34 of the ECD to Lys-101 of the TSH β-chain. A direct contact was observed in two homology models of the TSHR-ECD·TSH complex (one selected model depicted) supporting the feasibility of a contribution to specific TSH binding. F, superposition of the TSHR-LRR domain (white, PDB code 2xwt) at the C-terminal region with the FSHR-ECD/FSH complex (PDB code 4ay9). The reduced curvature of the TSHR-LRR resulted in an increased distance to the hormone in the N-terminal part of the TSHR template. G, distances between the cross-linked residues Lys-45 of the GPH α-chain and Thr-66 in the ECD to which the hormone is bound (green lines) and to the ECD of the adjacent ECD-hormone complex (red lines).

FIGURE 4.

Double mutant studies of the TSHR. G_s signaling of the TSHR and variants in the absence and presence of bTSH. The S281C/I486C and S281D/I486D double mutants do not show ligand-induced cAMP accumulation (A). In the case of the S281C/I486C double mutant, the transition to an activated receptor conformation is most likely hindered by a disulfide bond introduced between the two residues. The radioligand binding assay of the TSHR and variants (B) shows that the S281C/I486C variant is still capable of ligand binding, suggesting that the missing ligand induced receptor activation is caused by a disruption of the activation process. mU, milliunits.

FIGURE 5.

Structural variability at the ECD/TMD interface in homology models of the TSHR. Shown is the superposition of the best scoring homology models of the largest clusters for the TSHR-ECD (A) and the TSHR-TMD (B). The ECD models are structurally similar at the putative TMD interface located at the terminal α-helix excluding the connecting loop (depicted in orange), which was removed before docking. The models of the TMD, in contrast, show greater variations in the putative interface at the extracellular loops (light orange, ECL1; yellow, ECL2; white, ECL3).

FIGURE 6.

Evaluation of docking results by interface score and Ser-281/Ile-486 Cβ distance. Two areas (black rectangles) in the plot of interface score (dG separated) versus Cβ distance between Ser-281 and Ile-486 (A) were selected for reconstruction of the connecting loop. The best scoring models (dG separated <−6) with a Ser-281/Ile-486 Cβ distance <10 Å (B) show a broad variety of ECD/TMD orientation with a few clusters of similar orientations (up to 4 structures). Structures scoring significantly better (dG separated <−9) with a Ser-281/Ile-486 Cβ distance between 10 and 15 Å (C) display a funnel at a Ser-281/Ile-486 Cβ distance of 12.5 Å with almost all models displaying a similar ECD/TMD orientation.

FIGURE 7.

Full-length models of the TSHR. A, the best scoring full-length TSHR model of cluster one after remodeling of the connecting loop between ECD and TMD shows an almost upright orientation of the ECD toward the membrane. B, the conformation of ECL1 includes an extended transmembrane helix 3 in the largest cluster with Ile-486 facing away from the interface with the ECD, resulting in an increased Ser-281/Ile-486 Cβ distance. C, in the best model of cluster four only a small helical fragment was retained within the loop, resulting in closer proximity of Ser-281 and Ile-486. Thr-490 is located in the extended TM3 and might influence the putative transition between the extended TM3 and the loop conformation of this region during receptor activation.

FIGURE 8.

Analysis of full-length receptor models after reconstruction of the connecting loop. Comparison of the score versus Cβ distance of Ser-281 and Ile-486 after clustering (A) shows that the best models based on score and cluster size display a Ser-281/Ile-486 Cβ distance >10 Å. Differences in contact maps (B) of cluster one and four (blue, contact in every model of cluster one and in none of cluster four; red, contact only in cluster four) for the Ser-281/ECL1 interface showing that the Ser-281/Ile-486 contact is only observed in cluster four (upper black rectangle) and the aromatic environment of Ser-281 including Tyr-481and Tyr-279 is only observed in cluster one (lower black rectangle). Superposition of the best scoring structure of cluster four (green) and the β2-adrenergic receptor (white, PDB code 2rh1) with the side chains of the WXFG motif depicted (C) shows that ECL1 of the homology model adopts a similar loop conformation in this region.