Fig. 1.

A, two Cys bridges (Cys¹⁴-Cys²⁰⁰ and Cys¹¹⁴-Cys¹⁹⁶) and two ionic pairs (Arg³⁸-Asp⁹⁸ and Glu⁹⁰-Lys¹²¹) in homology model of human GnRH receptor in the inactive conformation. Inactive (gray) and active (yellow) states were modeled using rhodopsin crystal structures (Protein Data Bank codes 1U19 and 3DQB, respectively). EL2 loops (residues 181–204) are colored blue, Cys residues are colored orange, Arg and Lys residues are colored blue, and Asp and Glu residues are colored red. Superposition of active and inactive conformation shows that the largest conformational changes include outward rigid body movement of TM6, movement of TM5 toward TM6, tilting of TM7, and shift of its middle part toward TM2. B and C, the proposed movement of the EL2 in the wild-type and mutant human GnRH receptor. B, homology models of the wild-type human receptor (yellow) in active state with GnRH decapeptide (purple) show the upper position of the EL2 (blue), which is attached to the receptor by two disulfide bridges (Cys¹⁴-Cys²⁰⁰ and Cys¹¹⁴-Cys¹⁹⁶). C, homology model of the ΔLys¹⁹¹, L112F, Q208E, L300V, and D302E mutant of the human receptor in the inactive state (gray) with EL2 loop (blue) inserted between helices and filling the ligand binding pocket. Deletion of charged Lys¹⁹¹ permits the movement of EL2 inside the seven-helical bundle. Substitutions in noncontiguous four-residue motif that interacts with EL2 (Gln²⁰⁸ forms H-bond with His¹⁹⁹, Val³⁰⁰ allows reorientation of Tyr²⁹⁰ that interacts with His¹⁹⁹, and Glu³⁰² forms H-bond with Gln¹⁹⁵) may facilitate the inward movement of EL2. Multiple interaction of EL2 with helices may stabilize the receptor structure. D to F, rearrangement of ionic bridges and H-bond networks during activation of wild-type and mutant human GnRH receptor. D, model of the inactive conformation of receptor in complex with peptide antagonist cetrorelix. Two ionic bridges are formed between TM1-TM2 (Arg³⁸-Asp⁹⁸) and TM2-TM3 (Glu⁹⁰-Lys¹²¹). The H-bond network that is formed in TM1-TM3-TM6-TM7 (Asn⁵³-Asn⁸⁷-Asp³¹⁹-Asp³¹⁵-Trp²⁸⁰) additionally stabilizes the receptor structure. Antagonist interacts with Arg³⁸-Asp⁹⁸ salt bridge, but not with Lys¹²¹. E, model of the active conformation of the receptor complexed with the natural decapeptide agonist GnRH. The ionic bridge Glu⁹⁰-Lys¹²¹ is broken, and Lys¹²¹ together with Asp⁹⁸, interact with His² of GnRH. Arg³⁸ forms H-bond with Gly¹⁰-NH₂ of the ligand. Rearranged residues (Asn⁵³-Ser⁴⁹ and Asn⁸⁷-Glu⁹⁰-Asn³¹⁵-Glu³¹⁹-Ser²⁷⁵) form H-bond network in TM1-TM2-TM3-TM6-TM7. Tyr²⁸⁴ rotates and forms H-bond with Ser²¹⁷. F, model of the E90K mutant of human GnRH receptor in complex with Asp²-GnRH decapeptide, which acts as an agonist. Salt bridge Glu⁹⁰-Lys¹²¹ is broken, and Lys¹²¹ interacts with Asp² of the ligand and also forms an ionic pair with Asp⁹⁸. H-bond network is similar to one in the active conformation of the wild-type receptor. Lys⁹⁰ is involved not only in H-bonding with Asn³¹⁵ but also in the ionic attraction with Asp³¹⁹ and repulsion with Lys¹²¹. These ionic and H-bonding interactions of Lys⁹⁰ probably stabilize the active conformation, which leads to CA. Interaction of decapeptide ligands (colored purple) in all three models are in good agreement with the experimental data.