Bringing GPCR Structural Biology to Medical Applications: Insights from Both V2 Vasopressin and Mu-Opioid Receptors

Abstract

G-protein coupled receptors (GPCRs) are versatile signaling proteins that regulate key physiological processes in response to a wide variety of extracellular stimuli. The last decade has seen a revolution in the structural biology of clinically important GPCRs. Indeed, the improvement in molecular and biochemical methods to study GPCRs and their transducer complexes, together with advances in cryo-electron microscopy, NMR development, and progress in molecular dynamic simulations, have led to a better understanding of their regulation by ligands of different efficacy and bias. This has also renewed a great interest in GPCR drug discovery, such as finding biased ligands that can either promote or not promote specific regulations. In this review, we focus on two therapeutically relevant GPCR targets, the V2 vasopressin receptor (V2R) and the mu-opioid receptor (µOR), to shed light on the recent structural biology studies and show the impact of this integrative approach on the determination of new potential clinical effective compounds.

Keywords: G protein-coupled receptor; Nuclear Magnetic Resonance; X-ray crystallography; arginine-vasopressin receptor; cryo-electron microscopy; molecular dynamics; μ-opioid receptor.

Figure 1

Schematic representation of GPCR activation. Prior to ligand binding, the receptor is in an apo-state (receptor is in white). Upon the binding of an antagonist (red circle) to a receptor, there is no activation (receptor is in red). Upon the binding of an agonist (green circle), the receptor is in the pre-activation state (receptor is in yellow), and the G protein heterotrimer binds to the active receptor (active receptor is in green). The exchange of GDP for GTP in the G protein α subunit leads to dissociation and interaction with downstream effectors such as the Gα subunit with the adenylyl cyclase (AC) and Gβγ subunits that activate ion channels. Pre-activated receptors can also signal through arrestins. The phosphorylation of the receptor C-terminal tail (yellow circle) by G protein-coupled receptor kinase (GRK) binding (active receptor is in green) promotes arrestin recruitment (active receptor is in green), which can internalize the phosphorylated receptor. Gray dashed circles highlight the common binding pocket of the receptor where the α5-helix (blue cartoon) of the Gα subunit and the finger loop (purple cartoon) of arrestin bind upon the activation of the receptor (grey cartoon). The G proteins, GRK and arrestin are shown in blue, pink and purple, respectively. The apo, inactive, agonist-bound and active states of the receptor are shown in white, red, yellow, and green, respectively.

Figure 2

Comparison of the pros and cons of the main structural biology methods.

Figure 3

Timeline of major advancements in the GPCR field. The achievements of X-ray crystallography, structure-based drug design and cryo-EM are shown in blue (light blue for XFEL), orange, and green, respectively.

Figure 4

Close-up view of the µOR binding site. µOR structures are shown as gray cartoon representations, D^3.32 and W^6.48 are displayed as green stick representations. (a) β-FNA is in violet (4dkl); (b) DAMGO is in light yellow (6dde) and light brown (8efq); (c) oliceridine is in dark green (8efb); (d) PZM21 is in blue (7sbf) and pink (8efo) and FH210 is in orange (7scg); (e) BU72 is in raspberry (5c1m) with morphine in green (8ef6); (f) MP is in brown (7t2g: 2 poses). (g) fentanyl in blue (8ef5), SR17018 in cyan (8efl) and LFT in light green (7t2h); (h) C5 guano is in blue (7u2l), and C6 guano is in gold (7u2k).

Figure 5

Structures of µOR in different states. (a) X-ray structures of the inactive-(left) and active-state (right) µOR bound to antagonist β-FNA and agonist BU72, respectively. (b) Cryo-EM structure of µOR in complex with the DAMGO and Gi protein. All proteins are shown as cartoon representations and ligands are not shown. µOR are depicted in a green color range. Gαi subunit is shown in orange, Gβ subunit is shown in cyan, Gγ subunit is shown in purple. BRIL, Nb39 and ScFv16 are illustrated in pink, yellow and light blue, respectively. The PDB file accession numbers are indicated for each complex.

Figure 6

Structures of V2R complexes. (a) Cryo-EM structures of V2R with Gs protein. (b) Cryo-EM structures of V2R with β-arrestin1. All proteins are shown as cartoon representations and AVP are not shown. V2R are depicted in the blue color range. The Gαs subunit is shown in brown (mini Gαs in 7kh0 and 7dw9, complete Gαs in 7bb6), the Gβ subunit is shown in cyan, and the Gγ subunit is shown in purple. Nb35 and ScFv16 are illustrated in gray and light blue, respectively. β-arrestin1 and ScFv30 are shown in pink and pale cyan, respectively. The PDB file accession numbers are indicated for each complex.