Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor

Abstract

FSH, a glycoprotein hormone, and the FSH receptor (FSHR), a G protein-coupled receptor, play central roles in human reproduction. We report the crystal structure of FSH in complex with the entire extracellular domain of FSHR (FSHR(ED)), including the enigmatic hinge region that is responsible for signal specificity. Surprisingly, the hinge region does not form a separate structural unit as widely anticipated but is part of the integral structure of FSHR(ED). In addition to the known hormone-binding site, FSHR(ED) provides interaction sites with the hormone: a sulfotyrosine (sTyr) site in the hinge region consistent with previous studies and a potential exosite resulting from putative receptor trimerization. Our structure, in comparison to others, suggests FSHR interacts with its ligand in two steps: ligand recruitment followed by sTyr recognition. FSH first binds to the high-affinity hormone-binding subdomain of FSHR and reshapes the ligand conformation to form a sTyr-binding pocket. FSHR then inserts its sTyr (i.e., sulfated Tyr335) into the FSH nascent pocket, eventually leading to receptor activation.

Fig. 1.

Structure of human FSH in complex with human FSHR_ED. (A) Ribbon model of human FSH bound to FSHR_ED: FSH α-subunit is shown in green, FSH β-subunit is shown in purple, and FSHR_ED is shown in magenta. The side chain of sulfated Y335 is depicted as sticks for the tyrosine stem and colored balls for the sulfate group. The carbohydrates are depicted as balls. The disordered residues in the receptors are marked as dashed lines. (B) Top view of the trimer observed in the asymmetrical unit.

Fig. 2.

Structure of FSHR_ED. (A) Schematic diagram of the topology of the FSHR structure. The regular secondary structure elements of LRRs are shown as arrows for the β-strands and as a cylinder for the α-helix. Disulfide bonds are shown as yellow lines linking cysteines, the N-linked glycosylation sites are shown as “Y,” and the exon boundaries in the FSHR gene are shown as black triangles (▼). (B) Ribbon representation of FSHR_ED. The hormone-binding subdomain is shown in yellow, and the signal specificity subdomain (hinge region) is shown in magenta. (Inset) In the close-up view of the signal specificity subdomain, motif CF3 is colored green, the hormone-binding region is colored yellow, and the rhodopsin-like extracellular sequence is colored magenta. The disulfide bonds are shown as brown sticks, sulfated Y335 is shown as sticks for the phenyl ring and balls for the sulfate group, and the side chain of S273 is shown as balls. As a reference, FSH is included as a gray surface.

Fig. 3.

Hormone-receptor interactions outside the primary binding site. (A) sTyr site, with close-up views of the interactions within the FSH–FSHR_ED complex monomer (Upper Inset) and the electrostatic potential of the sulfated tyrosine-binding pocket (Lower Inset). (B) Potential exosite related to trimerization, with a close-up view (Inset) of the interactions originating from the FSH–FSHR_ED complex oligomerizations. The receptor trimer is shown as a magenta surface. FSH is shown as ribbons: green ribbons for the α-chain and light purple ribbons for the β-chain. The FSH N52α glycan is shown as yellow balls.

Fig. 4.

Formation of the sulfate-binding pocket in FSH at the sTyr site. (A and B) Comparison of the ℒ2β loop of FSH in free form (red wire) and FSHR_ED-bound form (brown wire). FSH α-subunit is shown as green ribbons, and the rest of the β-subunit is shown as blue ribbons. Sulfated FSHR Y335 is shown as balls as a reference. The orientation of FSH in A and B is related by a rotation of 60°. Residues around A43β in the ℒ2β loop of FSH swing ∼10 Å from the free form to the FSHR_ED-bound form to form the sulfate-binding pocket. (B, Inset) Newly formed FSH pocket is structurally supported by the hydrophobic interactions between the left wall of the pocket and the FSHR residues around V221 and I222. (C and D) Surface representations show the rearrangement of the FSH ℒ2β loop on receptor binding. FSH α- and β-subunits are colored green and blue, respectively.

Fig. 5.

Two-step FSH/FSHR recognition and functional validations of the sTyr site and the exosite. (A) Schematic diagram of the two-step recognition. The GPHR’s extracellular LRRs, in a putative orientation relative to the seven-transmembrane (7TM) domain, are shown as magenta blocks with the flexible loop as a hairpin, and the 7TM domain is shown as cylinders with the inactivated state colored gray and the activated state colored green. The hormone-binding subdomain is labeled as HBSD, and signal specificity subdomain is labeled as SSSD. Sulfated Y335 is shown as yellow balls, residue S271 is shown as green stars, and disulfide bonds are shown as brown sticks. Heterotrimeric G_s or β-arrestin protein is shown as a green ellipsoid. GPH heterodimer is represented in purple, whereas carbohydrates at N52α are shown as a yellow Y-shaped stick. Shown here is only one of the possible orientations of the extracellular domain relative to the 7TM domain. (B) Importance of electrostatic potential of FSH in lifting the Y335-harboring FSHR hairpin for receptor activation. Negative potential is colored red, and positive potential is colored blue. FSHR_ED is represented as magenta ribbons, with the side chain of sulfated Y335 shown as sticks. (C) Validation of the roles of the sTyr site and the exosite in FSHR activation by FSH mutagenesis. (Left) Relative amount of β-arrestin recruited for binding to the activated FSHR inside the CHO cell on stimulation by FSH or its mutants. The amount of recruited β-arrestin is normalized to 100% for the maximum response of FSH. Data represent experiments performed with quadruplicate samples. (Right) Relative amount of estradiol production inside primary granulosa cells from immature rats on stimulation by FSH or its mutants. The amount of estradiol production is normalized to 100% for the maximum response of FSH. Data represent experiments performed with triplicate samples.