Structure and Dynamics of Helix-8 in GPCR-PDZ-Domain Interactions
The interaction of GPCRs with PDZ-domain-containing proteins is essential for signaling. Helix-8 (H8) is a structurally conserved amphipathic helical motif in class-A GPCRs, adjacent to the C-terminal sequence that is responsible for PDZ-domain-recognition. To understand the role of H8 in molecular level mechanisms of GPCR/PDZ interactions we investigated the interaction of the PDZ-containing GIPC1 protein with the dopamine D2 receptor (D2R) in homology models of the systems based on the X-ray structures of very closely related analogs: the D3R, and the PDZ domain of GIPC2, respectively. The 5 C-terminal residues of D2 receptor's relatively short C-terminal stretch next to H8 was docked into the GIPC1-PDZ model using a simulated-annealing-based PDZ docking scheme. On the resulting GIPC1-D2R complex we carried out several regular MD simulations in sphingolipid/cholesterol membranes to determine the stability of the complex. The results show that the free carboxyl group of C-terminus of the D2R preferred to sample the space around the main-chain atoms of the conserved GFGL motif located on the carboxylate-binding loop of the GIPC1-PDZ. We investigated the relation between distance of H8 from the membrane and its secondary structure by metadynamics MD simulations and found that as the H8 moved away from the membrane, it became energetically easier for the helix to unravel into an extended conformation that is the canonical conformation for interaction with PDZ-domain. Because the C-termini of GPCRs are palmitoylated, we also investigated effect of palmitoylation on the structure and dynamics of H8 in the context of its role in repositioning H8 for the interaction with the PDZ domain. We find that in the palmitoylated C-terminus the H8 backbone penetrates deeper into the membrane, whereas de-palmitoylation renders it more accessible to the cytoplasm where it can interact with the PDZ domain-containing proteins.