Abstract
The arginine-vasopressin (AVP) V2 receptor (V2R) is a G protein-coupled receptor that controls body water homeostasis. It is involved in many water balance and urine disorders. Point mutations of its gene are directly responsible for two rare genetic diseases. As such, it is a key therapeutic target. Despite important progress in understanding the molecular basis of its function, it remained for a long time refractory to structure determination. This work is thus focused on the determination of the three-dimensional (3D) V2R structure in complex with its canonical signaling partners Gs protein or βarrestin1 (βarr1) by cryo-electron microscopy (Cryo-EM). The comparison of the two active states of the V2R at an atomic level is an important step toward the understanding of the molecular mechanisms involved in its activity.We first successfully determined the AVP-V2R-Gs complex structure by using a combination of Cryo-EM, experimental NMR, and molecular dynamic simulations. This structural biology hybrid approach allowed to solve molecular details of AVP binding to V2R and of the interface of the receptor with the Gs protein signaling partner. The structure is in agreement with molecular pharmacology data accumulated over 20 years. The binding pocket is a deep cleft in the center of the seven-helix bundle. The bottom of the orthosteric crevice is mainly composed of hydrophobic residues while the entrance is more hydrophilic. The active V2R displays hallmarks of receptor activation such as a large outward movement of the transmembrane domain (TM)6 an inward movement of the TM7 and a break of the Ionic lock involving helices TM3 and TM6 (D/ERY motif). The coupling between the receptor and its Gs signaling partner is significantly tighter compared to what is observed for other class A GPCRs and interestingly, strongly dynamic, allowing us to characterize three conformational sub-states. This study goes further than a simple description of a receptor or a signaling protein complex structure. Indeed, 3D models were interpreted to understand the structural consequences of V2R mutations responsible for two rare genetic diseases. Congenital Nephrogenic Diabetes Insipidus (cNDI) is associated with V2R loss-of-function mutations whereas Nephrogenic Syndrome of Inappropriate antidiuresis (NSIAD) is associated with V2R constitutively active mutations.