Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

Abstract

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Extended Data Fig. 1. Constitutively active rhodopsin interacts with arrestin and GαCT

a. SDS-PAGE of N-terminally MBP-tagged WT and mutant rhodopsin. b. Non-cropped versions of the pull-down assay gels shown in Fig. 1B. The interactions between mouse WT arrestin and human WT or E113^3.28Q rhodopsin are very weak. In contrast, the interaction between constitutively active rhodopsin (E113^3.28Q /M257^6.40Y) and pre-activated L374A/V375A/F376A arrestin (3A_arrestin) is strong and is further increased in the presence of 10 μM all-trans retinal. Input: 5% of the binding reaction. Bottom panels show the rhodopsin loading controls. c, Schematic representation of the AlphaScreen assay. d. AlphaScreen binding assay between E113^3.28Q /M257^6.40Y rhodopsin and GαCT (TGGRVLEDLKSCGLF) in the presence and absence of 5 μM all-trans retinal. The two left columns show the controls with “peptide only” and “rhodopsin only”. (n=3, error bars=SD). e. Determination of the affinity of the interaction between rhodopsin E113^3.28Q /M257^6.40Y and GαCT by homologous competition. His6 -MBP-rhodopsin mutant protein was immobilized on Ni-acceptor beads and biotinylated GαCT on streptavidin donor beads. Binding between rhodopsin and arrestin brings donor and acceptor beads into close proximity, resulting in the indicated binding signal. Non-biotinylated GαCT competed for the interaction with an IC₅₀ of ~700 nM (n=3, error bars=SD).

Extended Data Fig. 2. Purification and crystallization of T4L-rhodopsin-arrestin

a, Purification of the T4L-rhodopsin-arrestin (T4L-Rho-Arr) complex. His8-MBP-MBP-T4L-Rho-Arr complex was first purified by amylose column chromatography (lane1). The His8-MBP-MBP tandem tag was then released by cleavage with 3C protease (lane 2) and removed by binding to Ni-NTA beads to recover pure T4L-rhodopsin-arrestin (T4L-Rho-Arr) protein (lane 3). b, Analytical gel filtration profile of the T4L-rhodopsin-arrestin complex. T4L-rhodopsin-arrestin eluted mostly as monomers with a small proportion of oligomers. The molecular weights of protein standards are indicated at the top. c, Thermal stability shift analysis of T4L-rhodopsin-arrestin. T4L-rhodopsin-arrestin is relatively stable with a T_m of 59 °C. d, Crystals of T4L-rhodopsin-arrestin in lipidic cubic phase under bright field illumination (top) and polarized light (bottom). e, X-ray diffraction pattern of a T4L-rhodopsin-arrestin crystal recorded at LS-CAT of APS. The green ring indicates the position of reflections at 8.0 Å resolution.

Extended Data Fig. 3. Electron density map for the overall complex and the key interfaces based on the XFEL data

a, A 2F_o–F_c electron density map contoured at 1 σ of the arrestin finger loop, which forms the key interface with TM7 and Helix 8. b, A 2F_o-F_c electron density map contoured at 1 σ of the loop between TM5 and TM6, which forms the key interface with the β-strand following the finger loop. c, A 3000K simulated annealing omit map (2F_o-F_c electron density map contoured at 1 σ) calculated from the 3.8Å/3.8Å/ 3.3Å XFEL data supports the overall arrangement of the rhodopsin-arrestin complex. In all panels, the complex structure is shown with rhodopsin colored in green, arrestin in brown and T4L in yellow. f, Stereo views of the lariat loop with a 2F_o-F_c composite omit map at 1 σ calculated from the 3.8Å/3.8Å/ 3.3Å truncated XFEL data. Key residues are labeled.

Extended Data Fig. 4. Structure similarity of the four rhodopsin-arrestin complexes in the asymmetric units and the interface between rhodopsin and arrestin

a, Two 90° views of the superposition of the four rhodopsin-arrestin complexes are shown as cartoon representation. The four complexes have an RMSD of less than 0.5 Å in the Cα atoms of rhodopsin and arrestin. b, Close-up view of arrestin-binding sites in rhodopsin. The four arrestin-binding sites (P1–P4) are highlighted in brown on the rhodopsin surface. The rhodopsin C-terminal tail/arrestin interface (P4) is based on computational modeling and disulfide crosslinking data. c, Rhodopsin-binding sites in arrestin. The four rhodopsin-binding sites (P1-P4) are highlighted in green on the arrestin surface.

Extended Data Fig. 5. Conformational modeling of the rhodopsin-arrestin full length complex

a, An overview of the computational model. b, Predicted interactions of the rhodopsin C-terminus with arrestin, showing strong to medium pairwise restraints between Cβ atoms of rhodopsin and arrestin residues identified by disulfide crosslinking. c, same as in b, but showing predicted hydrogen bonding and ionic interactions for the C-terminal residues of rhodopsin.

Extended Data Fig. 6. Dynamics of free 3A_arrestin and rhodopsin-bound arrestin determined by HDX

a, HDX perturbation map between rhodopsin-bound arrestin and free arrestin, which is derived from the difference in the HDX rate between rhodopsin-bound arrestin and free arrestin. The bars below the arrestin sequence represent the peptide fragments resolved by mass spectrometry and the colors of the bars indicate the relative decrease in deuterium exchange (color code at bottom). b, The thermal stability of free 3A_arrestin and the rhodopsin-arrestin complex shows that the rhodopsin-arrestin complex is more stable than free 3A_arrestin.

Extended Data Fig. 7. Cell based Tango assays to validate the rhodopsin-arrestin interface

a, Cartoon illustration of the Tango assay for rhodopsin-arrestin interactions in cells. b–c, Mutations of key arrestin (b) and rhodopsin (c) residues that mediate the rhodopsin-arrestin interactions. Tango assay were performed in the absence or presence of 10 μM all-trans-retinal (ATR). (n=3, error bars=SD).

Extended Data Fig. 8. Control experiments for disulfide bond cross-linking specificity

a, The product of the cross-linking reaction of finger loop residue G77C with N310^7.57C of TM7 was confirmed by western blots using anti-FLAG antibody (which detects arrestin-FLAG fusion) and anti-HA antibody (which detects rhodopsin-HA fusion). The cross-linked products are marked with arrow heads, and free-arrestin and free-rhodopsin are indicated by asterisks. Arrestin (3A) and rhodopsin (4M) without cysteine mutations do not form cross-linked products. b, The cross-linked product of finger loop residue G77C with N310^7.57C of TM7 was sensitive to treatment with reducing agents, indicating the cross-linking is mediated through disulfide bond formation. c, A close-up view of arrestin finger loop residues M76C and G77C and their cross-linking with rhodopsin, which shows that G77C was specifically cross-linked to N310^7.57C of TM7 and Q312^8.49 of Helix 8, and M76C was cross-linked to N310^7.57C of TM7 and Q312^8.49C of Helix 8, but not to other residues. d, Structure and cross-linking of finger loop N-terminal residues Q70C, E71C, and D72C of arrestin to T70C and K67C from ICL1 of rhodopsin. e, Structure and cross-linking of arrestin back loop residues R319C and T320C to Q237^ICL3C from TM5 of rhodopsin.

Extended Data Fig. 9. Structure comparison of the arrestin-bound rhodopsin with the β₂-adrenergic receptor in complex with G_s protein (PDB code: 3SN6) and the inactive rhodopsin (PDB code: 1F88)

a, Superposition of arrestin-bound rhodopsin (green) with G_s protein-bound β₂ adrenergic receptor (light yellow). The major conformational changes are indicated by arrows. b, An intracellular view of a superposition of arrestin-bound rhodopsin (green) and G_s protein-bound β₂-adrenergic receptor (light yellow). c, Overlays of arrestin-bound rhodopsin (green) with inactive rhodopsin (pink) reveals specific conformational changes in each TM helix. The arrows indicate outward movements of TM helices. d, RMSD of Cα atom differences between arrestin-bound rhodopsin and inactive rhodopsin shows the large conformational changes in TM5 and TM6.

Extended Data Fig. 10. Structure of rhodopsin-bound arrestin and its comparison with inactive and “pre-activated” arrestin

a–b, The charge potential surface map of rhodopsin from the rhodopsin-arrestin bound complex shows that the cytoplasmic rhodopsin TM bundle surface is positively charged (blue) whereas its C-terminal tail is negatively charged (red). c–d, Charged surface of arrestin from the rhodopsin-arrestin bound complex shows that the arrestin finger loop is negatively charged (red) and its N-terminal β-strand interface is positively charged (blue). The charge distribution in rhodopsin and arrestin is complementary to each other for their interactions. e, Comparison of rhodopsin-bound arrestin (light blue) with inactive arrestin (brown, PDB code: 1CF1), showing an ~20° rotation between the N- and C- domains of arrestin. f, Comparison of rhodopsin-bound arrestin (dark brown) with pre-activated arrestin (light brown, PDB code: 4J2Q), showing conformational changes in the finger loop, which adopts an α-helical conformation (cyan) in the complex. The extended finger loop conformation would protrude into the rhodopsin TM bundle and is not compatible with receptor binding. Computational model for the full rhodopsin-arrestin complex is shown in panels (b) and (d).

Extended Data Fig. 11. A computational model of phosphorylated rhodopsin in complex with arrestin and salt sensitivity of the rhodopsin-arrestin interaction

a–d, An overall view (a) and close-up views (b–d) of the computational model of the rhodopsin C-tail with phospho-serine at positions 334, 338 and 343 in complex with arrestin. e, The AlphaScreen control (biotin-His6) shows much less salt sensitivity than the interaction between His-tag-rhodopsin and biotin arrestin, which is very sensitive to salt, with an IC₅₀ of around 200 mM NaCl (100 mM NaCl added to 100 mM salt of the original assay buffer) (n=3, error bars=SD).

Extended Data Fig. 12. A positive charge property is commonly found at the cytoplasmic side of GPCRs

a–e, Surface charge potential of the cytoplasmic side of selected agonist bound GPCR structures: β₁AR, PDB code: 2Y02 (a); β₂AR, PDB code: 3PDS (b); A_2A adenosine receptor, PDB code: 3QAK (c); serotonin receptor 5HT_1B, PDB code: 4IAR (d); serotonin receptor 5HT_2B, PDB code: 4IB4 (e). Positive and negative charge potentials are shown in blue and red, respectively. f, Sequence alignment of the finger loop region highlighting negatively charged residues (shown in red), which are conserved in all subtypes of arrestins.

Extended Data Fig. 13. A possible role of the arrestin C-edge in lipid binding

a,b The asymmetric assembly of the rhodopsin-arrestin complex in the presence of a lipid membrane bilayer, showing the C-edge of arrestin dipping into the lipid layer. c,d, A close-up view of the C-edge of arrestin in the membrane layer, where the conserved hydrophobic side chains are shown. The figure was made using the computational model for the full rhodopsin-arrestin complex.

Figure 1. Rhodopsin-arrestin interactions and complex assembly

a, Diagram of the binding of rhodopsin (Rho) with G-protein and arrestin as described in the main text. Labels are 11-cis retinal (ECR) and all-trans-retinal (ATR). b, Rhodopsin and arrestin interaction determined by pull-down assay in the absence and presence of ATR (top panel). Middle panel: rhodopsin loading controls. Bottom panel: Relative binding of ³⁵S-labelled arrestin was determined by densitometry (n=3, error bars=SD). c, Binding of His8-MBP-rhodopsin (E113^3.28Q /M257^6.40Y) protein to biotin-MBP-arrestin (WT and 3A) measured by AlphaScreen in the absence or presence of 5 μM ATR. The first six columns are controls (luminescence signals in the presence of only one of the binding partners; n=3, error bars=SD). d, Competition of arrestin binding to rhodopsin was determined by a homologous AlphaScreen assay and the IC₅₀ value was derived from repeat experiments (n=3, error bars=SD). e, Negative stain EM images of rhodopsin-arrestin complexes without or with T4L at the N-terminus; Right panel: Overlay of the EM image with the structures of T4L, rhodopsin and arrestin. m: detergent micelle.

Figure 2. The structure of the rhodopsin-arrestin complex

a, The structure of the rhodopsin-arrestin complex in four orientations. The relative dimensions of rhodopsin and arrestin are shown in the intracellular view. TM1-TM7 indicates rhodopsin transmembrane helices 1–7; H8 is intracellular Helix 8. b, An overall view of the rhodopsin-arrestin complex shown with transparent solid surface. T4 Lysozyme (T4L) is omitted from this view. c, Crystal packing diagram of the rhodopsin-arrestin complex with T4L as yellow ribbon model.

Figure 3. DEER validation of rhodopsin-arrestin complex assembly

a, An overall view of rhodopsin-arrestin assembly showing the three intermolecular distances based on the models of the R1 nitroxide pairs at rhodopsin residue Y74^2.41 and three arrestin residues T61, V140, and S241 based on the crystal structure. b–d, The experimental distance distributions between the nitroxide spin labeled R1 pairs of rhodopsin Y74^2.41 and bovine arrestin S60, V139, and L240, which are in equivalent positions to mouse arrestin T61(b), V140 (c), and S241(d) as labeled in the figure. Y axes are the probability of distance distribution.

Figure 4. The rhodopsin-arrestin interface and its validation by HDX

a–b, Two overall views showing the four interface patches of the rhodopsin-arrestin complex. c–d, Mapping of HDX on the rhodopsin-bound arrestin structure. Rhodopsin is colored in red and arrestin is colored based on the exchange rate differences between free 3A_arrestin and rhodopsin-bound arrestin as shown in Extended Data Fig. 6a. This figure was made using a computational model of the full rhodopsin-arrestin complex.

Figure 5. Validation of the rhodopsin-arrestin interface by disulfide bond cross-linking

a–e, Structure and cross-linking of arrestin with rhodopsin. Panels are arrestin finger loop with rhodopsin TM7 and helix 8 (a); arrestin middle loop with rhodopsin ICL1 (b); arrestin lariat loop residue Y251 with rhodopsin TM5 (c); arrestin β-strand interface residues with residues of rhodopsin TM5, ICL3, and TM6 (d); and arrestin’s N-terminus with rhodopsin’s C-tail (e). Rhodopsin K311 is marked with a red star and arrestin M76 is show in full for clarity. Part of the computational model for the full rhodopsin-arrestin complex was used in panel (e).

Figure 6. Structural basis of arrestin-biased signaling and arrestin recruitment

a–b, Two views of structural overlays of arrestin-bound rhodopsin (green) with inactive rhodopsin (pink). c–d, Two views of structural overlays of arrestin-bound rhodopsin (green) with GαCT peptide-bound rhodopsin (orange). e, A cartoon model of arrestin recruitment by a phosphorylated and active rhodopsin. In the dark state, the receptor is inactive (R-state) and arrestin is in the closed state (basal state). Receptor activation and phosphorylation (P-R* state) allow the phosphorylated C-terminal tail of rhodopsin to bind to the N-domain of arrestin (pre-activated state), thus displacing the arrestin C-terminal tail. This displacement destabilizes the polar core of arrestin, which allows a 20° rotation between the arrestin N- and C- domains, leading to the opening of the middle loop (ML) and lariat loop (LL) to accommodate the ICL2 helix of rhodopsin (fully-activated state). The activated receptor also opens the cytoplasmic side of the TM bundle to adopt the finger loop (FL) of arrestin.