Conformational selection guides β-arrestin recruitment at a biased G protein-coupled receptor

Abstract

G protein-coupled receptors (GPCRs) recruit β-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage β-arrestins but not G proteins, making them a model system for investigating the structural basis of β-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on ¹³CH₃-ε-methionine-labeled ACKR3, revealing that β-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular β-arrestin-coupling region. NMR studies of ACKR3 mutants defective in β-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides β-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Fig. 1.. β-arrestin-biased signaling at ACKR3 and structural characterization by NMR.

(A) CXCL12 activates β-arrestin but not G protein at ACKR3. (B) CXCL12-mediated cAMP inhibition of CXCR4 (positive control) and ACKR3 in Glosensor assay (EC₅₀ = 0.21 nM, CXCR4) (top). CXCL12-mediated β-arrestin-2 recruitment to ACKR3 in Tango assay (EC₅₀ = 3.9 nM, ACKR3) (bottom). N=3 in triplicate in both assays; error bars reflect S.E.M. (C) β-arrestin recruitment to ACKR3 via Nano-BiT assay. VUN701 dose response alone could not be fit (black circles). Purple circles reflect VUN701 dose response with CXCL12 at 3.2 nM. See text and Table S1 for EC₅₀ and E_max. All conditions N=3 in duplicate. Error bars reflect S.E.M. (D) Summary of ligand potency at β-arrestin recruitment. (Structures not to scale; CXCL12 PDB: 2KEC; nanobody from PDB 6KNM used to represent VUN701; LIH383 and CCX777 were modeled in PyMol). LIH383 sequence is FGGFMRRK (20). The chemical structure of CCX777 is shown in (19). (E) ¹H-¹³C HSQC NMR spectra of WT-ACKR3 with various ligands at 310 K. Assigned methionine residues are labeled; (*) denotes inferred assignments from other ligand-bound states (Methods). Negative contour peaks shown in semi-transparency and dashed lines. Peaks marked “a” encompass natural abundance peaks from buffer and detergent components (see also Fig. S2G, S3). All spectra are shown at the same contour except LIH383, which was lowered to represent M212^5×39.

Fig. 2.. Conformational changes in the ligand binding pocket and intracellular region characterize the β-arrestin recruiting state.

(A) ACKR3 model depicting M212^5×39 and M138^3×46 probes. (B) Overlay of M212^5×39 peaks from ligand-bound ACKR3 complexes at 310K. 16–16.5 and 18.5–19 p.p.m. peaks (¹³C) correspond to gauche and trans rotameric states, respectively. Ligand-specific β-arrestin activity is depicted at right. Open and closed locks depict conformational sampling and restriction, respectively (bottom). (C) Ligand-residue interactions were compared between antagonist and β-arrestin-biased ligand-bound GPCRs (Methods). Comparison of the mean number of ligand contacts with TM5, ECL2, and TM7 residues. * p-value < 0.05; ** p-value < 0.005 (unpaired t-test). (D) AT₁R contacts with the ligand TRV023 (β-arrestin-biased; black outline) in TM5, ECL2, and TM7 shown as blue spheres (PDB 6OS1). The position of NMR probe 5×39 in AT₁R is shown. Stabilization of TM5-ECL2-TM7 by biased ligands depicted as a “lock” (right). (E) Met138^3×46 peaks in all four ligand-bound states. Upfield peak positions (¹H: ~1.3 p.p.m.) of M138^3×46 among agonist-bound states supports ring-current shifts due aromatic side chain interactions. Peaks marked “a” as in Fig. 1. (F) 3×46-contacts residues at 2×43 and 7×53 exclusively in active state complexes and 6×37 exclusively in inactive-state complexes (Methods). (G) β-arrestin recruitment of WT ACKR3 versus alanine mutants of 3×46-contacting mutants by NanoBiT (N = 3). See also Table S1.

Fig. 3.. Mutational inactivation of β-arrestin recruitment through the conserved polar network.

(A) Position of Asn127^3×35 in ACKR3 model relative to Met212^5×39 and Met138^3×46. (B) β-arrestin-2 recruitment with CXCL12 for ACKR3 mutants Asn127Lys^3×35 and Asn127Ser^3×35 in Tango assay. All conditions N=3 in triplicate. Error bars reflect S.E.M. See also Table S1. (C) Closeup of ¹H-¹³C-HSQC in Met212^5×39 region (right), highlighting VUN701-WT-ACKR3 (first panel), CXCL12-ACKR3 Asn127Lys^3×35 (second panel), CXCL12-wild type (WT)-ACKR3 (third panel), and CXCL12-ACKR3 Asn127Ser^3×35 (fourth panel). (D) Overlay of ¹³C-HSQC in the Met138^3×46 region for WT-ACKR3-VUN701, ACKR3-Asn127Lys^3×35-CXCL12, ACKR3-Asn127Ser^3×35-CXCL12, and WT-ACKR3-CXCL12 complexes. A shaded triangle suggests peak non-collinearity. Peaks marked “a” as in Fig. 1. See S8A for Met138^3×46 assignment method. (E) Normalized CSPs for Met212^5×39 in ¹³C-dimension (y-axis) and Met138^3×46 in ¹H-dimension (x-axis) from random coil for Asn127^3×35 mutants and CXCL12- and VUN701-bound WT-ACKR3. Arrows depict transitions among CXCL12-bound WT- and mutant ACKR3. (F) Depiction of differences between WT-ACKR3 and Asn127Lys^3×35 in CXCL12-bound states. Despite being bound to CXCL12 (blue key), Asn127Lys^3×35 “locks” ACKR3 in the inactive state, abrogating CXCL12’s effects on Met212^5×39 and Met138^3×46 probes. (G) Comparison of residue-residue interactions AT₁R-β-arrestin biased ligand and -antagonist bound states (top) reveals formation of a 3×39–7×46 interaction in the β-arrestin state (bottom).

Fig. 4.. Dynamic control of β-arrestin recruitment to ACKR3.

Allosteric regulation of GPCR β-arrestin activation by coordinated transitions in conformational heterogeneity. Ligands constrain (agonists) or promote (antagonists) conformational heterogeneity by altering intermolecular (ligand-residue) and intramolecular (residue-residue) interactions throughout the GPCR structure.