Research resource: novel structural insights bridge gaps in glycoprotein hormone receptor analyses

Abstract

The first version of a glycoprotein hormone receptor (GPHR) information resource was designed to link functional with structural GPHR information, in order to support sequence-structure-function analysis of the LH, FSH, and TSH receptors (http://ssfa-gphr.de). However, structural information on a binding- and signaling-sensitive extracellular fragment (∼100 residues), the hinge region, had been lacking. A new FSHR crystal structure of the hormone-bound extracellular domain has recently been solved. The structure comprises the leucine-rich repeat domain and most parts of the hinge region. We have not only integrated the new FSHR/FSH structure and the derived homology models of TSHR/TSH, LHCGR/CG, and LHCGR/LH into our web-based information resource, but have additionally provided novel tools to analyze the advanced structural features, with the common characteristics and distinctions between GPHRs, in a more precise manner. The hinge region with its second hormone-binding site allows us to assign functional data to the new structural features between hormone and receptor, such as binding details of a sulfated tyrosine (conserved throughout the GPHRs) extending into a pocket of the hormone. We have also implemented a protein interface analysis tool that enables the identification and visualization of extracellular contact points between interaction partners. This provides a starting point for comparing the binding patterns of GPHRs. Together with the mutagenesis data stored in the database, this will help to decipher the essential residues for ligand recognition and the molecular mechanisms of signal transduction, extending from the extracellular hormone-binding site toward the intracellular G protein-binding sites.

Figure 1.

Schematic Illustration of GPHR Topology and a Homology Model of hLHCGR N-ECR with Bound Lutropin. A, GPHRs possess a 7-TMH topology (serpentine domain consists of 7-TMHs connected by 3 extracellular loops [ECLs] and 3 intercellular loops [ICLs]), as do all GPCRs. In contrast to most family A GPCRs, they have a very large N-ECR. This N-ECR is composed of the LRRD and the hinge region. The LRRD is made up of 11 full repeats. Cb-1 comprises a cysteine from the N terminus, 2 cysteines from an antiparallel β-strand upstream and one from the first regular repeat. The hinge region, now structurally defined by the new FSHR crystal structure, starts after a short helix-element of the 11th repeat and contains the first 2 cysteines of Cb-2, which are linked via disulfide bridges to Cb-3. After the helix, an unstructured region harbors the cysteine box 2/3 linker and the cleavable peptide (C-peptide), in the case of TSHR. The sulfotyrosine is known from mutagenesis studies and contributes to hormone binding in all 3 GPHR subtypes. The C-terminal section of the hinge region contains a β-strand that is located parallel to the last repeat of the LRRD and is connected via a disulfide bridge. B, Homology model of the N-ECR of hLHCGR in complex with hLH. The gray ribbon presentation visualizes the Cα atom backbone of the LRRD with 9 repeats, according to previous information concerning the LRRD dimension. The new structural information for the LRRD (33) is colored magenta and includes an extension of the LRRD by repeat 11 (β-strand [light green] and a short helix [light blue]), a coiled region with the sulfated tyrosine 331 (blue sticks) and finally a β-strand orientated parallel to the last strand of the LRRD. The 2 cysteines of the short helix are bridged via disulfide bonds between the β-strand and the C terminus of the hinge region. The hLH is visualized as a surface (α-subunit in pale orange, β-subunit in pale green).

Figure 2.

Protein-Protein Interface Analysis Tool. The display shows the homology model of hTSHR (LRRD in gray; hinge region in magenta) in complex with the hormone bTSH (α-subunit in orange, β-subunit in green) as visualized at the SSFA-GPHR website. The surface of the hormone is colored according to the receptor-hormone contact proximity of the van der Waals radius between the interaction partners. Red spots are the closest contacts and usually correspond to hydrogen bonds. Yellow and green spots highlight more distant side chains and often correspond to hydrophobic interactions. As an example, in panel A, all negatively charged N-ECR residues in close contact to the hormone are visualized together with the contact surface displayed on bTSH. The inset shows a close-up of particular negatively charged side chains of the hinge region interacting with the hormone. In panel B the interaction partners (positively charged side chains) on the interface are highlighted (labeled manually for representation).