Valine-279 Deletion-Mutation on Arginine Vasopressin Receptor 2 Causes Obstruction in G-Protein Binding Site: A Clinical Nephrogenic Diabetes Insipidus Case and Its Sub-Molecular Pathogenic Analysis.

Congenital nephrogenic diabetes insipidus (CNDI) is a genetic disorder caused by mutations in arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 genes, rendering collecting duct cells insensitive to the peptide hormone arginine vasopressin stimulation for water reabsorption. This study reports a first identified AVPR2 mutation in Taiwan and demonstrates our effort to understand the pathogenesis caused by applying computational structural analysis tools. The CNDI condition of an 8-month-old male patient was confirmed according to symptoms, family history, and DNA sequence analysis. The patient was identified to have a valine 279 deletion-mutation in the AVPR2 gene. Cellular experiments using mutant protein transfected cells revealed that mutated AVPR2 is expressed successfully in cells and localized on cell surfaces. We further analyzed the pathogenesis of the mutation at sub-molecular levels via long-term molecular dynamics (MD) simulations and structural analysis. The MD simulations showed while the structure of the extracellular ligand-binding domain remains unchanged, the mutation alters the direction of dynamic motion of AVPR2 transmembrane helix 6 toward the center of the G-protein binding site, obstructing the binding of G-protein, thus likely disabling downstream signaling. This study demonstrated that the computational approaches can be powerful tools for obtaining valuable information on the pathogenesis induced by mutations in G-protein-coupled receptors. These methods can also be helpful in providing clues on potential therapeutic strategies for CNDI.

Internal water channel formation in CXCR4 is crucial for Gi-protein coupling upon activation by CXCL12.

Chemokine receptor CXCR4 is a major drug target for numerous diseases because of its involvement in the regulation of cell migration and the developmental process. In this study, atomic-level molecular dynamics simulations were used to determine the activation mechanism and internal water formation of CXCR4 in complex with chemokine CXCL12 and Gi-protein. The results indicated that CXCL12-bound CXCR4 underwent transmembrane 6 (TM6) outward movement and a decrease in tyrosine toggle switch by eliciting the breakage of hydrophobic layer to form a continuous internal water channel. In the GDP-bound Gαi-protein state, the rotation and translation of the α5-helix of Gαi-protein toward the cytoplasmic pocket of CXCR4 induced an increase in interdomain distance for GDP leaving. Finally, an internal water channel formation model was proposed based on our simulations for CXCL12-bound CXCR4 in complex with Gαi-protein upon activation for downstream signaling. This model could be useful in anticancer drug development.

Activation-Induced Conformational Changes of Dopamine D3 Receptor Promote the Formation of the Internal Water Channel.

The atomic-level dopamine activation mechanism for transmitting extracellular ligand binding events through transmembrane helices to the cytoplasmic G protein remains unclear. In the present study, the complete dopamine D3 receptor (D3R), with a homology-modeled N-terminus, was constructed to dock different ligands to simulate conformational alterations in the receptor's active and inactive forms during microsecond-timescale molecular dynamic simulations. In agonist-bound systems, the D3R N-terminus formed a "lid-like" structure and lay flat on the binding site opening, whereas in antagonist and inverse agonist-bound systems, the N-terminus exposed the binding cavity. Receptor activation was characterized using the different molecular switch residue distances, and G protein-binding site volumes. A continuous water pathway was observed only in the dopamine-Gαi-bound system. In the inactive D3Rs, water entry was hindered by the hydrophobic layers. Finally, a complete activation mechanism of D3R was proposed. Upon agonist binding, the "lid-like" conformation of the N-terminus induces a series of molecular switches to increase the volume of the D3R cytoplasmic binding part for G protein association. Meanwhile, water enters the transmembrane region inducing molecular switches to assist in opening the hydrophobic layers to form a continuous water channel, which is crucial for maintaining a fully active conformation for signal transduction.