Figure 1.
The FSHR LRRD and hinge region with bound FSH.
The crystal structure of the FSHR LRRD/hinge region fragment/FSH complex is trimeric [54] (insert, boxed window). A monomeric LRRD/FSH/hinge region complex was extracted from the trimeric arrangement to visualize specific important structural and functional features. The LRRD was previously assumed to end at LRR9 with an additional tenth β-strand [21], [23]. The new structure shows an additional eleventh repeat and extends the LRRD through amino acid Asn280 (green backbone). This is followed by a short helical secondary structure, comprising the first two cysteines (Cys275, Cys276, yellow lines) of cysteine box 2 (Cb-2, N-terminal hinge region). These form disulfide bridges with cysteines (Cys346, Cys356, yellow) of cysteine-box 3 (Cb-3, C-terminal hinge region, chocolate brown), in which a short β-strand is additionally assembled parallel to the eleventh strand of the concave LRRD β-sheet. The N-terminal section of the hinge region after the LRRD is crystallized till residue Ser295. The inner cysteine bridge (Cys292-Cys338) between the two remaining extracellular cysteines connects this section with the C-terminal hinge region. It was already known from mutagenesis studies that the sulfated Tyr335 (at the C-terminal hinge region that is crystallized from position Thr331-Ile359) is mandatory for hormone binding and signaling and interacts tightly with amino acids of the hormone subunits, between the β-subunit loop 2 and the α-loop 1. The side chains of the hormone constitute a binding pocket for the sulfated tyrosine; light green sticks represent aromatic residues, green sticks hydrophobic residues and orange sticks hydrophilic residues. The adjacent negatively charged Asp334 interacts directly with the positively charged Arg53 of the hormone β-subunit. The C-terminus of the hinge region at Cys346 forms a β-strand parallel to the last repeat strand of the LRRD and ends with residues that are conserved among the GPHRs and that are known from studies on TSHR to be relevant to signaling [11], [44].
Figure 2.
Amino acid sequences of hormone subtypes and specific receptor fragments.
A) Alignment of hormone subtype amino acid sequences with signal peptides (numbering with signal peptides throughout the manuscript). The α-subunits of hTSH, bTSH, hTSH analogue (TR1401, Trophogen Inc., Rockville, MD) and thyrostimulin are shown, together with the β-subunit sequences of hFSH, hLH and hCG. Residues are colored according to their biophysical properties: blue - positively charged, red - negatively charged, green - hydrophobic, orange - hydrophilic, magenta – histidine, brown – cysteine, black – proline, light green - aromatic residues). B) Comparison of the amino acid sequences of different GPHR subtypes and LGRs. Residues are presented from the cysteine-box 2 through the transmembrane helix 1. Specificities are highlighted, including the positions of CAMs and amino acids relevant to binding.
Figure 3.
The TSHR LRRD and hinge region in complex with TSH.
A-B) Homology model of the extracellular TSHR region complexed with bTSH. The new LRRD (pale beige backbone ribbon) repeat LRR 11 (green) contains a helical secondary element with two consecutive cysteines (yellow) of cysteine-box 2. The hormone is visualized as a surface (α-subunit in orange, β-subunit in greenblue). The sulfated tyrosine 385 (TSHR number, red stick) [49] location is comparable to that in the FSHR/FSH complex interacting with the hormone in a pocket constituted by hydrophilic (orange sticks), aromatic (light green sticks) and hydrophobic residues (green sticks). In contrast to FSH and human TSH, bovine TSH contains additional positively charged residues at the α-subunit (blue surface regions in A) and blue sticks at B)) which are known to increase hormone binding properties compared to hormone variants without those positive charges at the corresponding positions [70]. Our TSHR model suggests that these four side chains (at the α-subunit interact with negatively charged asparagines and glutamates (red sticks) in the TSHR hinge region (Asp382, Glu303) that have been shown experimentally to interplay with positively charged hormone side chains of bTSH [51], [52]. These interactions are responsible for the super-agonistic effects of bTSH compared to hTSH and are perfectly matched in this TSHR/TSH model based on the FSHR/FSH complex. Met300 and Leu299 (hydrophobic, green surface and sticks) and the negatively charged Glu297 (red stick in B) are already known to be sensitive to TSHR binding; these also have complementary partners on the hormone (details in B). Moreover, Cb-2 and C-b3 interact tightly. Amino acids at the N- and C- terminal sites (magenta) close to TMH1 (the serpentine domain) have been shown by mutation studies to activate TSHR constitutively. In Cb-2 (A), Pro280 and Ser281 at the end of helical repeat 11 region are in direct spatial proximity to amino acids Pro400–Pro407 of Cb-3. One difference between the β-subunits of bTSH and hTSH is not directly explained by the current TSHR/TSH model. The positively charged βArg89 of bTSH is absent in hTSH and substitution of this residue into hTSH leads to greatly enhanced binding of hTSH to TSHR [70], [71]. The TSHR monomer model reveals that this residue is located at the β-L3 loop and is not involved in any intra- or intermolecular interaction to the targeted receptor.
Figure 4.
The extracellular TSHR region in complex with thyrostimulin and antibodies.
A) The second endogenous hormone-ligand for TSHR is thyrostimulin [63]–[66] (sequence at Figure 2). Structural details of a potential heterodimeric thyrostimulin have already been described [2], [80], [81]. On the assumption that the general orientation of heterodimeric thyrostimulin at the LRRD is similar to that for bTSH (Figure 3), thyrostimulin should also interact with the sulfated tyrosine in the C-terminal hinge region. In contrast to bTSH, two positively charged residues participate in the formation of the TSHR tyrosine 385 binding site (Figure 3B), which might influence the binding properties for the sulfated negative charged sulfated tyrosine. B) TSHR LRRD crystal structures are available with an interacting activating [23] or inactivating [22] antibody (AB), respectively. The activating M22 (light violet surface) and the inactivating (green backbone) K1–70 bAB are bound at the concave site of the LRRD like the hormone TSH (orange and blue backbone). Relative to K1–70, the activating AB is displaced at the LRRD towards the membrane spanning part. In this study, a new extended LRRD based on the FSHR LRRD crystal structure with an additional repeat (green backbone) was modeled and structurally aligned with the antibody/LRRD structures. It is observed that M22 could interact with more residues than previously assumed, e.g. at LRR10 and 11, including Arg274 (blue stick). The activating and inactivating antibodies contact LRRD in the same general region as the hormone, but the second binding site at the hinge region seems to be hormone specific. The orientations of the hinge fragments participating in hormone binding have been identified in an active/bound LRRD/hormone complex and might therefore be differently located in TSHR/antibody-complexes. The hormone-sensitive sulfated Tyr385 should probably contact the surface of M22, but is not tightly embedded in a specific pocket. Activating antibody/receptor contacts should be relevant to the displacement of specific hinge region fragments. The new TSHR model with hinge fragments complexed with M22 predicts very tight spatial proximity between this antibody and the signaling sensitive part (activation) of cysteine-box 3 (magenta sticks). This direct interface is not observable for TSH or for K1–70.
Figure 5.
Scheme of initial TSHR activation.
New functional-structural TSHR features at the extracellular site are described for the hinge region and the LRRD ain a schematic overview. Repeat 11 with the pivotal helix (green) is part of the LRRD that ends at Asn288 and therefore includes Cys283 and Cys284 of cysteine-box 2 (C-b2). These cysteines are linked by disulfide bridges to Cys398 and Cys408 of cysteine-box 3, respectively. Constitutively activating mutations at Pro280 and Ser281 (magenta) influence the conformation of this helix and underline the importance of this region for receptor function. Further positions of CAMs are located at the C-terminal part of C-b3, in close spatial proximity to Cb-2. Mutations at both fragments probably cause conformational changes at this region which induce receptor activation. Thus, these wild type amino acids constitute an activation-sensitive intramolecular agonistic unit (magenta, boxed). This unit is adjacent to TMH1 and is probably located between the ECLs as an interface between the extracellular and transmembrane regions. The transition between the extracellular region and TMH1 is made up of negatively charged amino acids, in which side chain substitutions lead to impaired signaling capacity. These are key to interactions with the serpentine domain spanning the membrane. Several residues identified as sensitive to hormone binding are enclosed in a red box (Glu297, Glu303, Asp382 and Asp386) and represent a spatial cluster that is linked by a disulfide bridge between Cys301 and Cys390. Asp382 (and Glu303) are important for super-agonistic effects of bTSH but are not important for hTSH. Sulfation at Tyr385 is obligatory for hormone binding and is accompanied by negatively charged amino acids close to Tyr385 (Asp386, Asp382). Two cleavage sites define the so-called cleavable peptide (C-peptide, ∼50 amino acids). Deletions at the transition between the C-peptide and the Cb-3 are reported to activate the TSHR (and also the FSHR) constitutively (magenta trapezoid). In contrast, single point mutations lead to partial inactivation of TSHR signaling at this region. The dashed arrows indicate the potential signal processing path for activation upon ligand binding from the hinge region and/or potentially via the LRRD. In either case, the extracellular modifications converge at the pivotal helix that links the intramolecular agonistic unit together (dashed box).