Figures and data
Cryo-EM reconstruction of the Gβγ–PLCβ3 Δ892-PHcys complex
(A) Domain diagram of human PLCβ3, with numbers above corresponding to domain boundaries. PLCβ3 is regulated by the X–Y linker (hot pink), proximal C-terminal domain (pCTD, cyan), and distal CTD (purple). The CTDs are connected by the unconserved CTD linker. (B) Cryo-EM density map and (C) structure of the 4 Å Gβγ–Δ892-PHcys complex crosslinked with BMOE, with PLCβ3 colored as in 1A, Gβ in blue, and Gγ in red.
Cryo-EM data collection, refinement and validation statistics
Functional analysis of Gβγ–PLCβ3 interfaces: IP accumulation.
Comparison of the (A) Gβγ–Δ892-PHcys interface observed in this study, and the previously reported (B) Gβγ–PLCβ3 PH and (C) Gβγ–PLCβ3 EF hand interfaces (PDB ID: 8EMW) (17). Proteins are colored as in Fig. 1. Residues in PLCβ3 and Gβγ shown as balls and sticks were mutated, and their basal and G protein-dependent activities quantified in a cell-based [3H]-IPx accumulation assay. Basal and Gαq-dependent activation was used as a control to confirm the PLCβ3 variants were properly folded. Changes in activity are not due to differences in expression (Figure S7). The activities of the PLCβ3 mutants in the (D) BMOE-crosslinked Gβγ–Δ892-PHcys interface and (E) Gβγ–PLCβ3 PH/EF hand interfaces were measured. Mutations to the Gβ1 subunit were similarly assessed for (F) the crosslinked Gβγ–Δ892-PHcys interface and (G) the Gβγ–PH/EF hand interfaces. All assays were performed in triplicate from at least three independent transfections, and data shown are mean ± SEM. Data in D and E were analyzed using a two-way ANOVA followed by Dunnett’s post-hoc multiple comparisons test, comparing the basal activity of each PLCβ3 variant to its activation Gβγ or Gαq. ***, p<0.0005, **, p<0.005, *, p<0.05. Data in F and G were analyzed using a one-way ANOVA, followed by Dunnett’s post-hoc multiple comparisons test, comparing each the Gβγ-stimulated activity of each PLCβ3 mutant to that of wild-type PLCβ3. ***, p<0.0005, **, p<0.005, *, p<0.05.
Functional analysis of Gβγ–PLCβ3 interfaces: interaction with G proteins and PI(4,5)P2 hydrolysis.
(A) BRET between membrane-anchored HiBit-PLCβ3-CAAX and Venus-Gβγ increases after activation of AT1 with angiotensin II (AngII; 1 μM). Signals are blocked by membrane-tethered GRK3ct (GRK3ct) and enhanced by membrane-tethered GRK2RH, which sequester free Gβγ and Gαq-GTP, respectively. Traces are the average of twenty replicates from five independent experiments. (B) AngII-induced changes in BRET between HiBit-PLCβ3-CAAX and Venus-Gβγ (ΔBRET) for sixteen mutants across the three Gβγ–PLCβ3 interfaces; most mutants showed significant changes (denoted by an asterisk) in their interactions with Venus-Gβγ compared to wild-type HiBit-PLCβ3-CAAX. (C) In ΔPLC cells, expression of HiBit-PLCβ3 (without LgBit) reconstitutes AngII-induced PI(4,5)P2 hydrolysis, as indicated by bystander BRET between Nluc-PH and mem-link-Venus (mem-Venus). Traces are the average of sixteen replicates from four independent experiments. (D) AngII-induced PI(4,5)P2 hydrolysis (ΔBRET) for the same HiBit-PLCβ3 mutants as panel B; most mutants showed significant changes in PI(4,5)P2 hydrolysis (denoted by asterisks). (E) In HEK cells, BRET between HiBit-PLCβ3 and Gαq-Venus increases after activation of AT1. Traces are the average of twelve replicates from three independent experiments. (F) BRET between the same HiBit-PLCβ3 mutants as panel B and Gαq-Venus; none of the mutants showed significant changes compared to wild-type HiBit-PLCβ3. For B, D, and F, all mutants were compared to wild-type HiBit-PLCβ3-CAAX or HiBit-PLCβ3 using one-way ANOVA with Dunnett’s post-hoc comparisons; data points represent averages from independent experiments (n=3-11) performed in quadruplicate. For all mutants, * p<0.05 and individual p values are given in SI Appendix, Tables S1-5.
Gβγ facilitation of PLCβ3 activation does not reflect membrane recruitment.
(A) Activation of AT1 induces translocation of HiBit-PLCβ3 to the plasma membrane as indicated by bystander BRET between HiBit-PLCβ3 and mem-Venus. Traces are the average of twelve replicates from three independent experiments. (B) BRET between the same HiBit-PLCβ3 mutants as Figure 3 and mem-Venus; none of the mutants showed a significant defect in membrane translocation compared to wild-type HiBit-PLCβ3. Individual p values are given in SI Appendix, Table S6. (C) In ΔPLC cells, expression of HiBit-PLCβ3-CAAX reconstitutes AngII-induced PI(4,5)P2 hydrolysis, indicated by bystander BRET between Nluc-PH and mem-Venus. Signals are inhibited by membrane-tetheredGRK3ct, which sequesters free Gβγ. Traces are the average of twenty-eight replicates from seven independent experiments. (D) PLCβ3-CAAX variants shown to be defective with respect to Gβγ binding are also defective with respect to PI(4,5)P2 hydrolysis. For B, all mutants were compared to wild-type HiBit-PLCβ3, and for D all mutants were compared to +GRK3ct RQ (a mutant defective in binding Gαq·GTP), using one-way ANOVA with Dunnett’s post-hoc comparisons; data points represent averages from independent experiments (n=3 or 7) performed in quadruplicate. For all mutants, * p<0.05 and individual p values are given in SI Appendix, Tables S6-7.
G protein–PLCβ3 complexes at the membrane.
(A) The crosslinked Gβγ–PLCβ3 complex is compatible with membrane localization, but not lipase activity. PLCβ3 is shown as a surface, and Gβγ in ribbon. Proteins are colored as in Figure 1A. (B) The liposome-tethered Gβγ–PLCβ3 complex would allow the PLCβ3 active site to interact with the membrane. (C) The crosslinked and (D) liposome-tethered complexes are compatible with Gαq·GTP binding and activation via displacement of the Hα2’ helix (cyan) and engagement of the dCTD (dark gray). In both models, the dCTD binds the membrane through electrostatic interactions.
Isolation of a Crosslinked and Functional Gβγ–PLCβ3 complex.
(A) Schematic of PLCβ3 Δ892 variants used for crosslinking studies. (B) Representative crosslinking experiments with Gβγ C68S and PLCβ3-Δ892 variants analyzed by SDS-PAGE and stained with Coomassie. PLCβ3-Δ892 undergoes extensive self-crosslinking (red box, left). Mutation of solvent-exposed cysteines and introduction of a single cysteine in the PH domain (E60C) eliminates self-crosslinking and allows crosslinking between Gβγ C68S and PLCβ3-Δ892PH (green box). PLCβ3-Δ892 XYcys, which lacks solvent-exposed cysteines except for C516 in the flexible X–Y linker, eliminated self-crosslinking but failed to crosslink to Gβγ-C68S. (C) BMOE-crosslinked complexes between wild-type Gβγ and PLCβ3 Δ892PH have higher activity in a liposome-based assay than the uncrosslinked control. Data are the mean of three separate experiments ± SD. Mock crosslinked and crosslinked samples were compared using an unpaired T-test. *, p < 0.05.
Cryo-EM densities of Gβγ–PLCβ3 Δ892-PHcys complexes.
(A) Top. Model of the larger particle population in the BMOE-crosslinked Gβγ–PLCβ3 Δ892-PHcys reconstruction fit into the cryo-EM density map. Bottom. Cryo-EM map colored by local resolution. (B) Model of the smaller particle population in the BMOE-crosslinked Gβγ–PLCβ3 Δ892-PHcys reconstruction fit into the cryo-EM density map. Bottom. Cryo-EM map colored by local resolution. (C) Top. Model of the BM(PEG)2-crosslinked Gβγ–PLCβ3 Δ892-PHcys reconstruction fit into the cryo-EM density map. Bottom. Cryo-EM map colored by local resolution. In all reconstructions, resolution is lower for Gβγ, consistent with a dynamic interface in solution.
Contoured cryo-EM density maps of the Gβγ–PLCβ3 Δ892-PHcys complexes.
The 4 Å Gβγ–PLCβ3 Δ892-PHcys complex fit in the density map contoured to (A) 0.197 σ or (B) 0.297 σ. The 7 Å Gβγ–PLCβ3 Δ892-PHcys complex contoured to (C) 0.197σ or (D) 0.227 σ, and the 4.5 Å BM-PEG crosslinked Gβγ–PLCβ3-Δ892 PHcys complex fit in density contoured to (D) 0.040 σ or (E) 0.050 σ.
Cryo-EM data workflow and resolution analysis of the BMOE-crosslinked Gβγ–PLCβ3 Δ892-PHcys complex.
The workflow, including a representative micrograph, 2D class averages (box size: 276 Å) and Fourier shell correlation (FSC) curves calculated from two independent reconstructions by CryoSPARC(58). The nominal resolution of the resulting map, as defined by the 0.143 cutoff, is indicated by the horizontal blue line.
Cryo-EM data workflow and resolution analysis of the BMPEG-crosslinked Gβγ–PLCβ3 Δ892-PHcys complex.
The workflow, including a representative micrograph, 2D class averages (box size: 276 Å) and Fourier shell correlation (FSC) curves calculated from two independent reconstructions by CryoSPARC(58). The nominal resolution of the resulting map, as defined by the 0.143 cutoff, is indicated by the horizontal blue line.
Gβγ–effector enzyme complexes.
Experimentally determined structures of Gβγ in complex with effector enzymes including (A) GPCR kinase 2 (GRK2, PDB ID 3CIK), (B) phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-Rex 1, PDB ID 6PCV), (C) liposome-tethered PLCβ3 (PDB ID 8EMW), (D) PLCβ3 in solution (this work, PDB ID 9Y7H), (E) the high affinity site of phosphatidylinositol 3-kinase γ (PI3Kγ, PDB ID 8SOC), and (F) both sites of PI3Kγ (PDB ID 8SOD). In all structures, Gβ is shown in dark blue, Gγ in navy, and the effector enzyme in gray. In the Gβγ–PI3K–Gβγ structures, the p110 subunit is shown in dark gray, and the p101 subunit in light gray.
Expression of PLCβ3 and Gβγ mutants.
Representative western blot image of cell lysates containing mutants of interest. (A) Western blot image of PLCβ3 mutants. (B) Western blot image of Gβ1 mutants. Transfected Gβγ is Avi-tagged (avi-Gβγ) and endogenous Gβ is detected in the untransfected controls. (C) Representative western blot image of cell lysates containing PLCβ3 and the R199A and R199E mutants (top) and co-transfected avi-Gβ and endogenous Gβ (below)
PLCβ binding surfaces are conserved in human Gβ1-4 isoforms.
Sequence alignment of human Gβ1-4 subunits. Residues highlighted in light gray are conserved in all isoforms and those highlighted in light green are share similar properties. Residues in orange and purple are those that bind only to the PLCβ3 PH domain or to EF hands, respectively, in the liposome-tethered reconstruction. Residues in green interact with PLCβ3 in both sites. Residues in cyan are unique to the crosslinked complex described in this work. Residues in dark blue interact with the PLCβ3 PH domain in the crosslinked and liposome-tethered reconstructions. Residues in hot pink are common to all three observed Gβγ-PLCβ3 interfaces. Asterisks correspond to residues known participate in other Gβγ–effector enzyme interactions. Sequences correspond to UNIPROT entries P62874 (Gβ1), P62879 (Gβ2), P16520 (Gβ3), and Q9HAV0 (Gβ4).
Gβγ binding residues are not well conserved across the human PLCβ isoforms.
Sequence alignment of the human PLCβ PH and EF hand domains. Conserved residues are highlighted in light gray and residues with similar properties are highlighted in light green. Residues in the PLCβ3 PH domain that bind Gβγ are in orange, and those that bind Gβγ through the EF hands are in purple. PLCβ3 residues that interact with Gβγ in the crosslinked structure are in cyan. Residues that interact with Gβγ in both cryo-EM structures are in dark blue. Sequences correspond to UNIPROT entries Q9NQ66 (PLCβ1), Q00722 (PLCβ2), Q01970 (PLCβ3), and Q15147 (PLCβ4).
Gαq binding promotes Gβγ binding to PLCβ3.
(A) BRET between membrane-anchored HiBit-PLCβ3-CAAX and Venus-Gβγ increases after activation of AT1 with angiotensin II (AngII; 1 μM) or dopamine D2R receptors with dopamine (DA; 100 μM). Traces represent the average of twenty replicates from five independent experiments. Signals were significantly smaller (p=0.0036) after activation of D2R; Welch’s t-test. (B) BRET between the Gβγ sensor memGRKct-Nluc and Venus-Gβγ increases after activation of AT1 or D2R. Traces represent the average of twenty replicates from five independent experiments. Signals were significantly larger (p=0.0008) after activation of D2R; Welch’s t-test. Transfection was identical for panels A and B with the substitution of memGRKct-Nluc for HiBit-PLCβ3-CAAX in panel B; Gαq and Gαi1 were overexpressed together with Venus-Gβγ in both panels. (C) BRET between HiBit-PLCβ3-CAAX wild-type (wt) and mutants with defective Gαq binding to the distal CTD (EEE) or proximal CTD (LE). Traces represent the average of 32 replicates from eight independent experiments. Signals were significantly smaller for both mutants; one-way ANOVA with Dunnett’s post-hoc comparisons.
PLCβ-mediated PI(4,5)P2 hydrolysis is facilitated by Gβγ derived from both Gq and Gi/o heterotrimers.
(A) In HEK cells, bystander BRET between Nluc-PH and mem-Venus decreases in response to AT1 activation with AngII (1 μM). Responses are inhibited by membrane-tetheredGRK3ct, which sequesters Gβγ, but not the binding-defective R587Q mutant (GRKct RQ), both before and after inactivation of Gi/o heterotrimers with pertussis toxin (PTX). Traces are the average twelve-sixteen replicates from 3-4 independent experiments. (B) Grouped data from the same experiments as panel A; indicated p values are from one-way ANOVA with Tukey’s multiple comparisons test. (C) In ΔPLC cells expressing PTX, HiBit-PLCβ3 L40E and R185E mutants fail to fully reconstitute AngII-induced PI(4,5)P2 hydrolysis compared to the wild-type (wt) enzyme; traces are the average of twelve replicates from three independent experiments. (D) Grouped data from the same experiments as panel C; indicated p values are from one-way ANOVA with Dunnett’s multiple comparisons test.
Expression of HiBit-PLCβ3 variants as indicated by LgBit-complemented luminescence in intact cells.
Expression of HiBit-PLCβ3-CAAX variants as indicated by LgBit-complemented luminescence in intact cells.
Angiotensin II-induced BRET between HiBit-PLCβ3-CAAX variants and Venus-Gβγ.
Angiotensin II-induced PI(4,5)P2 hydrolysis mediated by HiBit-PLCβ3 variants.
Angiotensin II-induced BRET between HiBit-PLCβ3 variants and Gαq-Venus.
Angiotensin II-induced BRET between HiBit-PLCβ3 variants and mem-Venus.
