Mechanism of Tethered Agonist Binding to an Adhesion G-Protein-Coupled Receptor

Abstract

Adhesion GPCRs (ADGRs) contain a GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the receptor N-terminus and undergoes autoproteolysis at a highly conserved GPCR proteolysis (GPS) site to generate the N-terminal fragment (NTF) and transmembrane C-terminal fragment (CTF). Dissociation of NTF reveals a peptide tethered agonist (TA) which is responsible for the activation of the ADGRs. The NTF-bound ADGRs contain the encrypted TA that assumes a β-strand configuration within the GAIN domain, which is markedly different from a U-shaped α-helical configuration of TA in the active cryo-EM structures of ADGRs. However, how the TA dramatically changes its configuration and binds to the ADGR CTF remains unknown. In this study, we have performed all-atom enhanced sampling simulations using a novel Peptide-Gaussian accelerated Molecular Dynamics (Pep-GaMD) method on TA binding to the ADGRD1. The Pep-GaMD simulations captured spontaneous binding of the TA into orthosteric pocket of the ADGRD1 and its large conformational transition from the extended β strand to the U-shaped α helical configuration. We were able to identify important low-energy conformations of the TA in the binding pathway, as well as different active and inactive states of ADGRD1, in the presence and absence of the Gs protein. Therefore, our Pep-GaMD simulations have revealed dynamic mechanism of the TA binding to an ADGR, which will facilitate rational design of peptide regulators of ADGRs.

Keywords: Adhesion G-protein-coupled receptors; Peptide-Gaussian accelerated Molecular Dynamics; binding; enhanced sampling; tethered agonist.

Figure 1.. Binding of tethered agonist (TA) in the G_S protein-coupled ADGRD1 receptor was observed in Pep-GaMD simulations:

(A) A representative binding pathway of the TA, which is colored by simulation time. (B) Time course of the fraction of native contacts between the TA and receptor calculated from five 2500 ns Pep-GaMD simulations. (C) Time course of the distance between the NE1 atom of W714^5.37 and O atom of L708^ECL2 calculated from five 2500 ns Pep-GaMD simulations. (D) 2D potential of mean force (PMF) free energy profile regarding the fraction of native contacts between the TA and receptor and W714^5.37-L708^ECL2 distance calculated by combining five 2500 ns Pep-GaMD simulations. The low-energy states are labeled as “Unbound/Open” (U/O), “Intermediate 1/Open” (I1/O), “Intermediate 2/Closed” (I2/C) and “Bound/Closed” (B/C).

Figure 2.. Dynamic motions of bound tethered agonist (TA) in the ADGRD1-G_S protein complex were observed in Pep-GaMD simulations:

(A) A representative structure of ADGRD1-G_S protein complex depicting the flexibility of bound TA. (B) Time course of the fraction of native contacts between the TA and receptor calculated from three 500 ns Pep-GaMD simulations. (C) Time course of the distance between the NE1 atom of W714^5.37 and O atom of L708^ECL2 calculated from three 500 ns Pep-GaMD simulations. (D) 2D potential of mean force (PMF) free energy profile regarding the fraction of native contacts between the TA and receptor and W714^5.37-L708^ECL2 distance calculated by combining three 500 ns Pep-GaMD simulations. The low-energy states are labeled as “Intermediate 2/Closed” (I2/C) and “Bound/Closed” (B/C).

Figure 3.. Low-energy conformational states of the tethered agonist (TA) binding in the ARGRD1-G_S complex:

(A-C) The “Intermediate 1/Open” (I1/O), “Intermediate 2/Closed” (I2/C) and “Bound/Closed” (B/C) states. The cryo-EM bound conformation of TA (PDB: 7WU2) is shown in grey as reference. (D) Critical residue interactions between TA (blue) and ADGRD1 (green) observed in the “I1/O” state. The TA formed polar and hydrophobic interactions with receptor residues H^1.35, N702^ECL2, W705^ECL2, S707^ECL2, S710^ECL2, W^5.37, F^5.39 and N781^ECL3. (E) Critical residue interactions between TA (blue) and ADGRD1 (orange) observed in the “I2/C” state. The TA formed polar and hydrophobic interactions with receptor residues Q^1.36, H^3.37, N703^ECL2, W705^ECL2, S707^ECL2, F^5.39, W^6.53, Y^7.39 and F^7.42. (F) Critical residue interactions between TA (blue) and ADGRD1 (grey) observed in the “B/C” state. The TA formed polar and hydrophobic interactions with receptor residues Q^1.36, N702^ECL2, W705^ECL2, A709^ECL2, W^5.37, N781^ECL3 and Q^7.40.

Figure 4.. Partial binding of tethered agonist (TA) in the ADGRD1 receptor without the G_S protein was observed in Pep-GaMD simulations:

(A) A representative partial binding pathway of the TA, which is colored by simulation time. (B) Time course of the fraction of native contacts between the TA and receptor calculated from five 1000 ns Pep-GaMD simulations. (C) Time course of the distance between the NE1 atom of W714^5.37 and O atom of L708^ECL2 calculated from five 1000 ns Pep-GaMD simulations. (D) 2D potential of mean force (PMF) free energy profile regarding the fraction of native contacts between the TA and receptor and W714^5.37-L708^ECL2 distance calculated by combining five 1000 ns Pep-GaMD simulations. The low-energy states are labeled as “Unbound/Closed” (U/C) and “Unbound/Open” (U/O).

Figure 5.. Inactivation of the ADGRD1 receptor was observed during only partial binding of tethered agonist (TA) without the G_S protein in Pep-GaMD simulations:

(A) The distance between the Cα atoms of V661^3.58 and K760^6.40 calculated from five 1000 ns Pep-GaMD simulations. (B) 2D potential of mean force (PMF) free energy profile regarding the fraction of native contacts between the TA and receptor and V661^3.58-K760^6.40 distance calculated by combining five 1000 ns Pep-GaMD simulations. The low-energy states are labeled as “Unbound/Active” (U/A) and “Unbound/Inactive” (U/IN). (C) The “Unbound/Inactive” (U/IN) conformation. The cryo-EM bound conformation of TA (PDB: 7WU2) is shown in grey as reference. (D) Comparison of the active cryo-EM conformation (grey) with the Inactive conformational state of the ADGRD1 receptor observed in the Pep-GaMD simulations.