Mechanism of beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy

Figures

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  Fig. 1. β-arrestin 1 (βarr1) uses two distinct sites to bind SH3. a, b Structures of βarr1 (green, PDB: 4JQI) and SH3 (magenta, PDB: 2PTK), cartoon representation. Residues used for disulfide trapping are shown as yellow sticks and labeled. Proline regions of βarr1 are framed in rectangles. c Heatmap of different SH3–βarr1 cysteine-pair complexes; densitometry analysis of Coomassie blue gels. The SH3–βarr1 complex band was normalized to the total density. Mean values from n = 3 independent experiments are shown; the gels were processed in parallel. d Top: representative results of disulfide trapping with βarr1_120C (binding site for SH3) and βarr1_116C (non-binding site for SH3) (Coomassie blue gel). Bottom: Western blot of disulfide trapping reaction between βarr1_120C and SH3_R95C confirms that the 60-kDa band is the SH3–βarr1 complex. Data are representative of n = 3 independent experiments (TCEP - Tris-(2-Carboxyethyl)phosphine, Hydrochloride).

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  Fig. 2. βarr1 binds the aromatic surface of SH3. a Cryo-EM density map of the SH3–βarr1-CC complex (green, βarr1; magenta, SH3; red, phosphopeptide mimicking the C-tail of vasopressin 2 receptor (V2Rpp); blue, Fab30; yellow, nanobody 32 (Nb32)). Map contour level is 0.75. b Interaction interface of SH3–βarr1-CC complex (green, βarr1; magenta, SH3) with density mesh (upsample map 0.69 Å/pix, contour level 0.37). Hydrogen bonds are shown as dashed lines. See also Supplementary Video 1. c Top panel: Cryo-EM density map of the SH3–βarr1-N complex (green, βarr1; magenta, SH3; red, V2Rpp; blue, Fab30). Map contour level is 0.50. Bottom panel: The interaction interface of SH3–βarr1-N complex (green, βarr1; magenta, SH3) with density mesh (upsample map 0.69 Å/pix, contour level 0.2). Only the backbone of SH3 was modeled; βarr1 side chains are shown for illustrative purposes. See also Supplementary Video 2. d Western blots of M2-FLAG pull-down assay of SH3-FLAG mutants and βarr1 wild-type (WT). Data are representative of n = 3 independent experiments. e Western blots of M2-FLAG pull-down assay of SH3-FLAG wild-type (WT) and βarr1 mutants. Data are representative of n = 3 independent experiments.

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  Fig. 3. HDX profile changes of free and V2Rpp-activated βarr1 in the presence of SH3. a Structures of free βarr1 (PDB: 1G4M) and V2Rpp-activated βarr1 (PDB: 4JQI, grey, βarr1; yellow, V2Rpp); regions with decreased and increased deuterium uptake are shaded in purple and pink, respectively. Only regions that exhibited statistically significant differences in deuterium uptake (> 0.3 Da for βarr1; >0.36 Da for βarr1–V2rpp) are indicated. Insets: focus on the βarr1-N and βarr1-CC sites. b HDX changes of βarr1 peptides corresponding to βarr1-CC and βarr1-N sites upon co-incubation with SH3 (mean ± SEM, n = 3 technical replicates; green, βarr1 or βarr1–V2Rpp alone; magenta, βarr1 or βarr1–V2Rpp in the presence of SH3). # D and % D – deuterium uptake, Da and %, respectively. Statistical analysis was performed using Welch’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). c Examples of peptides showing no changes in deuterium uptake upon co-incubation with SH3 (mean ± SEM, n = 3 technical replicates; green, βarr1 or βarr1–V2Rpp alone; magenta, βarr1 or βarr1–V2Rpp in the presence of SH3).

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  Fig. 4. βarr1 drives autoinhibition relief of Src. a Cryo-EM density map of the Src–βarr1-CC complex (green, βarr1; magenta, Src(SH3); red, V2Rpp; blue, Fab30; yellow, Nb32). Map contour level is 0.65. b The interaction interface of Src–βarr1-CC (green, βarr1; magenta, Src (SH3 is shown) with density mesh (upsample map 0.72 Å/pix, contour level 0.15). The hydrogen bonds are shown as dashed lines. See also Supplementary Fig. 8. c Cryo-EM density map of the Src–βarr1-CC complex low-pass filtered to 15 Å with the fitted model (green, βarr1; magenta, Src(SH3); red, V2Rpp; blue, Fab30; yellow, Nb32). Map contour level is 0.06. d βarr1 preferentially binds Src lacking C-tail phosphorylation at Y527. Left panel: schematic of the assay (green, βarr1; magenta, Src). Right panel, top: representative Western blot of Src phosphorylation by Csk (n = 4 independent experiments). Right panel, bottom: representative Western blot of M2-FLAG pull-down assay of βarr1-FLAG and C-tail phosphorylated and C-tail unphosphorylated Src (n = 8 independent experiments). e Schematic of βarr1-mediated activation of Src (green, βarr1; magenta, Src; orange, V2Rpp). See also Supplementary Video 3.

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  Fig. 5. N-domain of βarr1 interacts with SH1. a Schematic of cross-linking mass-spectrometry (CXMS) (green, βarr1; magenta, SH1). LC-MS - Liquid Chromatography-Mass Spectrometry. b Chemical cross-linking of purified SH1 and βarr1, Coomassie blue gels; data are representative of n = 3 independent experiments. The regions of the gels excised for in-gel trypsin digestion are framed in red rectangle. DSS - disuccinimidyl suberate, BS3 -Bis(sulfosuccinimidyl) suberate, DSP – dithiobis(succinimidylpropionate), DTSSP − 3,3´-dithiobis(sulfosuccinimidylpropionate), EGS - ethylene glycol bis(succinimidyl succinate). c Identified intramolecular cross-linking peptides (MaxQuant score cutoff =100). d Representative Western blot of GST pull-down assay of different constructs of GST-βarr1 (wild-type (WT), N-domain (1-175), C-domain (176-418)) and SH1 (n = 8 independent experiments). e Model of βarr1 (PDB: 1G4M) binding to the SH1 domain of Src (PDB: 1FMK) based on CXMS data (green, βarr1; magenta, SH1). The cross-link between K356 of SH1 and N-terminus of βarr1 is shown in yellow.

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  Fig. 6. SH3 binding induces distinct conformational changes in βarr1 and SH3. a–c Structural comparison of βarr1 in SH3–βarr1-CC (green), Src–βarr1-CC (salmon), inactive βarr1 (PDB: 1G4M, grey) and active βarr1 structures in complex with: chimeric β1AR with V2R tail (β1V2R) (PDB: 6TKO, yellow), chimeric M2 muscarinic receptor with V2R tail (M2V2R) (PDB: 6U1N, magenta), neurotensin receptor 1 (NTSR1) (PDB: 6UP7, cyan), V2Rpp–Fab30 (PDB: 4JQI, blue). a Focus on β-strand V. Left panel: superposition of inactive and active βarr1 structures, SH3–βarr1-CC, and Src–βarr1-CC. Right panel: residue offset in Src–βarr1-CC is shown in comparison with βarr1–V2Rpp–Fab30; the distance between Cα atoms of F75 is shown. b Left panel: β-strand V of the βarr1–V2Rpp–Fab30 (PDB: 4JQI, blue) does not fit into the Src–βarr1-CC density map. Right panel: the residue offset of β-strand V improves the fit into the density. The upsample map is used (0.72 Å/pix); map contour level is 0.1. c Focus on the middle loop. Left panel: superposition of inactive and active βarr1 structures and Src–βarr1-CC. Right panel: density around the middle loop in all respective structures (colouring is the same as in (a)). Structural…

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  Fig. 7. Both βarr1-CC and βarr1-N sites mediate Src activation in vitro and in cells. a Effect of βarr1 mutations on Src activity downstream of β2V2R. β2V2R, Src and βarr1 were transiently expressed in HEK-293 βarr1/βarr2 dKO cells. Representative Western blots and densitometry analysis of Src phosphorylation normalized to phospho-Src in control cells (no βarr1 transfected) (mean ±SEM; n = 6 biological replicates; one-way ANOVA with Dunnett’s post hoc test; **P < 0.01; blue, no βarr1; yellow, βarr1 wild-type (WT); red, βarr1 P88G_P91G; green, βarr1 P121E_P124G; cyan, βarr1 P121E_P124G; orange, βarr1 F80A). b Effect of βarr1 mutations on endogenous Src activity downstream of D1R. D1R and βarr1 were transiently expressed in HEK-293 βarr1/βarr2 dKO cells. Representative Western blots and densitometry analysis of Src phosphorylation normalized to phospho-Src in unstimulated cells (mean ±SEM; n = 4 biological replicates; one-way ANOVA with Dunnett’s post hoc test; **P < 0.01) βarr1 mutants numbering and colouring is the same as in (a). c Initial velocity of optimal Src peptide phosphorylation by Src (V0) alone or in the presence of βarr1 WT or βarr1 mutants activated by V2Rpp and Fab30. (mean…

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  Fig. 8. Excess of Src SH3 interferes with the core coupling of βarr1 to GPCRs and overexpression of Src affects desensitization of β2AR. a Structural superposition of Src–βarr1-CC (SH3, magenta; βarr1, green) with the β1V2R–βarr1 complex (β1V2R, slate; PDB: 6TKO), cartoon representation. The clashing region is indicated with the red arrow. b Core coupling of βarr1 finger loop bimane to phosphorylated V2R in membranes with and without SH3. Left panel: representative experiment of βarr1 finger loop bimane fluorescence. Right panel: peak fluorescence of βarr1 finger loop bimane (mean ±SEM; n = 6 independent experiments; one-way ANOVA with Dunnett’s post hoc test; *P < 0.05, ****P < 0.0001; grey, βarr1 alone; green, V2R(Apo)+βarr1; red, V2R + AVP+βarr1; yellow, V2R + AVP+βarr1 + SH3). c Core coupling of βarr1 finger loop bimane to the phosphorylated chimeric M2V2R reconstituted in lipid nanodiscs. Left panel: representative experiment of βarr1 finger loop bimane fluorescence. Right panel: peak fluorescence of βarr1 finger loop bimane (mean ± SEM; n = 3 independent experiments; one-way ANOVA with Dunnett’s post hoc test; **P < 0.01; grey, βarr1 alone; green, M2V2R(Apo)+βarr1; red,…