Mechanisms of peptide agonist dissociation and deactivation of adhesion G-protein-coupled receptors

Abstract

Adhesion G protein-coupled receptors (ADGRs) belong to Class B2 of GPCRs and are involved in a wide array of important physiological processes. ADGRs contain a GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the receptor N-terminus and undergoes autoproteolysis during biosynthesis to generate two fragments: the N-terminal fragment (NTF) and C-terminal fragment (CTF). Dissociation of NTF reveals a tethered agonist to activate CTF of ADGRs for G protein signaling. Synthetic peptides that mimic the tethered agonist can also activate the ADGRs. However, mechanisms of peptide agonist dissociation and deactivation of ADGRs remain poorly understood. In this study, we have performed all-atom enhanced sampling simulations using a novel Protein-Protein Interaction-Gaussian accelerated Molecular Dynamics (PPI-GaMD) method on the ADGRG2-IP15 and ADGRG1-P7 complexes. The PPI-GaMD simulations captured dissociation of the IP15 and P7 peptide agonists from their target receptors. We were able to identify important low-energy conformations of ADGRG2 and ADGRG1 in the active, intermediate, and inactive states, as well as exploring different states of the peptide agonists IP15 and P7 during dissociation. Therefore, our PPI-GaMD simulations have revealed dynamic mechanisms of peptide agonist dissociation and deactivation of ADGRG1 and ADGRG2, which will facilitate rational design of peptide regulators of the two receptors and other ADGRs.

Keywords: Protein-Protein Interaction-Gaussian accelerated Molecular Dynamics; adhesion G protein-coupled receptors; deactivation; enhanced sampling; peptide dissociation.

Figure 1.. Dissociation of the IP15 peptide agonist and deactivation of the human ADGRG2 receptor observed in the PPI-GaMD simulations:

(A) A representative dissociation pathway of the IP15 peptide agonist. (B) Root-mean-square deviations (RMSDs) of the IP15 peptide agonist relative to the cryo-EM bound conformation (PDB: 7WUI) calculated from the eight 1500 ns PPI-GaMD simulations. (C) The TM3-TM6 distance between the Cα atoms of L^3.58 and R^6.40 calculated from the eight 1500ns PPI-GaMD simulations. (D) 2D potential of mean force (PMF) free energy profile of the IP15 RMSD and L^3.58-R^6.40 distance calculated by combining the eight PPI-GaMD simulations. The low-energy states are labeled as “Bound/Active” (B/A), “Partially Dissociated 1/Intermediate 1” (PD1/I1), “Partially Dissociated 2/Intermediate 2” (PD2/I2), “Unbound/Active” (U/A), “Unbound/Intermediate 1” (U/I1), “Unbound/Intermediate 2” (U/I2) and “Unbound/Inactive” (U/IN).

Figure 2.. Low-energy conformational states of the IP15 peptide dissociation from the ARGRG2 receptor:

(A-B) The “Partially Dissociated 1/Intermediate 1” (PD1/I1) and “Partially Dissociated 2/Intermediate 2” (PD2/I2) states. The cryo-EM bound conformation (PDB: 7WUI) is shown in grey as reference. (C) The “Unbound/Inactive” (U/IN) state. (D) Critical interactions between IP15 (blue) and ADGRG2 (orange) observed in the (PD1/I1) state. The peptide agonist formed hydrogen-bonding (yellow dash lines) and hydrophobic interactions with receptor residues T1.43, D2.67, W2.69, F5.35, F769ECL2, P770ECL2, W779ECL2 and W6.60. (E) Critical interactions between IP15 (blue) and ADGRG2 (green) observed in the (PD2/I2) state. The peptide agonist formed mostly hydrophobic interactions with receptor residues L^3.40, F^5.35, Y^5.46, W^6.53 and F^6.57. (F) Unwinding of kink in TM6 observed in Intermediate “I1” (orange), Intermediate “I2” (green) and Inactive (red) states compared with the active cryo-EM conformation (grey).

Figure 3.. Dissociation of the P7 peptide agonist and deactivation of the human ADGRG1 receptor observed in the PPI-GaMD simulations:

(A) A representative dissociation pathway of the P7 peptide agonist. (B) Root-mean-square deviations (RMSDs) of the P7 peptide agonist relative to the cryo-EM bound conformation (PDB: 7SF8) calculated from the eight 1500 ns PPIGaMD simulations. (C) The TM3-TM6 distance between the Cα atoms of V^3.58 and L^6.40 calculated from the eight 1500ns PPI-GaMD simulations. (D) 2D potential of mean force (PMF) free energy profile of the P7 RMSD and V^3.58-L^6.40 distance calculated by combining the eight PPI-GaMD simulations. The low-energy states are labeled as “Bound/Active” (B/A), “Partially Dissociated 1/Intermediate 2” (PD1/I2), “Unbound/Active” (U/A), “Unbound/Intermediate 1” (U/I1), “Unbound/Intermediate 2” (U/I2) and “Unbound/Inactive” (U/IN).

Figure 4.. Low-energy conformational state of the P7 peptide dissociation from the ARGRG1 receptor:

(A) The “Partially Dissociated 1/Intermediate 2” (PD1/I2) state. The cryo-EM bound conformation (PDB: 7SF8) is shown in grey as reference. (B) The “Unbound/Inactive” (U/IN) state. (C) Critical interactions between P7 (blue) and ADGRG1 (green) observed in the (PD1/I2) state. The peptide agonist formed mostly hydrophobic interactions with receptor residues V549^ECL2, H550^ECL2, S^6.59, Q^7.35 and L^7.39. (D) Unwinding of kink in TM6 observed in Intermediate “I1” (orange), Intermediate “I2” (green) and Inactive (red) states compared with the active cryo-EM conformation (grey).