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Molecular basis for Gβγ-mediated activation of phosphoinositide 3-kinase γ

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gβγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ–human Gβγ complexes in the presence of substrates/analogs, revealing two Gβγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gβγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gβγ binding sites and interdomain contacts altered by Gβγ binding suggest that Gβγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gβγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ.

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Fig. 1: Cryo-EM reconstruction of the PI3Kγ·ADP–GβγHD–GβγCTD complex.
Fig. 2: Release of the tryptophan lock and α36 upon Gβγ binding to the p110γ-HD.
Fig. 3: Cryo-EM structure of PI3Kγ.
Fig. 4: Structural changes in PI3Kγ as a function of Gβγ binding.
Fig. 5: Functional characterization of Gβγ-mediated PI3Kγ activation.
Fig. 6: Proposed allosteric pathways in PI3Kγ.

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Data availability

All data needed to evaluate the conclusions in the paper are presented in the paper and/or Supplementary Information. Additional data related to this paper are available upon reasonable request from the authors. The structures of PI3Kγ, PI3Kγ·ATP, PI3Kγ·ADP, PI3Kγ·ADP–GβγHD, states 1 and 2 of PI3Kγ·ADP–GβγHD–GβγCTD, and their associated cryo-EM reconstructions have been deposited into the PDB under accession codes 8SO9, 8SOA, 8SOB, 8SOC, 8SOD and 8SOE, and the Electron Microscopy Data Bank under accession codes EMD-40650, EMD-40651, EMD-40652, EMD-40653, EMD-40654 and EMD-40655, respectively. Source data are provided with this paper.

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Acknowledgements

The authors acknowledge funding from NIH grants CA254402 (J.J.G.T.), CA221289 (J.J.G.T.), CA023168 (J.J.G.T.), HL071818 (J.J.G.T.), R35GM119787 (Q.D.), American Heart Association Postdoctoral fellowship (#834497, C.-L.C.), Purdue Center for Cancer Research (PCCR) Pilot Grant 2020-21 Cycle 2, PCCR Shared Resource 2020-2021 Cycle 1 (Grant #30001612), the Walther Cancer Foundation (J.J.G.T.) and the technical support of Purdue Life Sciences Electron Microscopy Facility for cryo-EM data collection.

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Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Chun-Liang Chen, Ramizah Syahirah, Sandeep K. Ravala, Yu-Chen Yen, Thomas Klose, Qing Deng & John J. G. Tesmer

  2. Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA

    Chun-Liang Chen, Sandeep K. Ravala, Yu-Chen Yen & John J. G. Tesmer

  3. Purdue Cryo-EM Facility, Purdue University, West Lafayette, IN, USA

    Thomas Klose

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Contributions

C.-L.C. and J.J.G.T. conceptualized the study. S.K.R. created the WT PI3Kγ construct, and C.-L.C. and Y.-C.Y. created the mutant PI3Kγ constructs. C.-L.C. produced and purified WT and mutant PI3Kγ proteins for conducting lipid kinase assays. Y.-C.Y. produced the WT Gβγ proteins. C.-L.C. and T.K. collected cryo-EM data, and C.-L.C. performed cryo-EM data analysis. C.-L.C. performed MDFF. C.-L.C. and J.J.G.T. performed atomic modeling for all the cryo-EM reconstructions of PI3Kγ without or bound with ligand, and Gβγ. R.S and Q.D. conducted the morpholino knockdown and rescue experiments using the zebrafish model system. C.-L.C. and J.J.G.T. wrote the original draft, and all authors further edited the manuscript.

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Correspondence to John J. G. Tesmer.

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Nature Structural & Molecular Biology thanks Glenn Masson, Suzanne Scarlata and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Workflow of cryo-EM data processing of PI3Kγ complexed with Gβγ and ADP.

The un-tilted (3525 frames, black scale bar = 100 nm) and 30° tilted datasets (2943 frames, scale bar = 100 nm) were collected and combined for data processing in CryoSPARC, resulting in 1103979 particles for further classification using RELION 3D classification with 3 classes. Particles in Class1 showing weaker GβγCTD density were further processed via CryoSPARC 3D classification. Most particles without the GβγCTD density classified into two species: PI3Kγ·ADP and PI3Kγ·ADP–GβγHD. The remaining particles containing the GβγCTD were combined with particles from RELION Classes 2 & 3, containing both GβγCTD and GβγHD, and subjected to masked 3D classification in CryoSPARC, resulting in two distinct conformations of PI3Kγ·ADP–GβγHD–GβγCTD, states 1 and 2, with GβγCTD and the associated p101 CTD exhibiting the largest variation.

Extended Data Fig. 2 Resolution estimation for cryo-EM reconstructions.

(A) PI3Kγ·ADP: 3.9 Å (B) PI3Kγ·ADP–GβγHD: 3.6 Å (C) State 1 of PI3Kγ·ADP–GβγHD–GβγCTD: 3.4 Å (D) State 2 of PI3Kγ·ADP–GβγHD–GβγCTD: 3.6 Å (E) PI3Kγ·ATP: 3.3 Å. FSC, Fourier shell correlation.

Extended Data Fig. 3 Cryo-EM reconstruction of PI3Kγ·ADP–GβγHD–GβγCTD in state1.

(A) Cryo-EM map of state 1 with domain colored and indicated in (M). (B) Close−up of the kinase domain in state 1 with no observable density for α36. (C) Kinase domain in the structure of PI3Kγ·ADP, with α36 indicated. (D) Gβγ residues involved in the p110γ-HD interaction. (E-F) Residues in the p110γ-HD–Gβγ interface. Residues are shown as sticks and colored according to atom type. (G-I) Cryo-EM map and model fitting for the p101-CTD–GβγCTD interaction. (J) Gβγ residues involved in the p110γ-CTD interaction. (K-L) Residues in the p101-CTD–GβγCTD interface. (M) Color key for domains in p110γ, p101, and Gβγ.

Extended Data Fig. 4 Cryo-EM reconstruction of PI3Kγ·ADP–GβγHD–GβγCTD in State 2.

(A) Cryo-EM map of state 2 with domain colored and indicated in (M). (B) Close-up of the kinase domain in state 2 with no observable density for α36. (C) Kinase domain in PI3Kγ·ADP, with α36 indicated. (D) Gβγ residues involved in the p110γ-HD interaction. (E-F) Residues in the p110γ-HD–Gβγ interface. Residues are shown as sticks and colored according to atom type. (G-I) Cryo-EM map and model fitting for the p101-CTD–GβγCTD interaction. (J) Gβγ residues involved in the p110γ-CTD interaction. (K-L) Residues in the p101-CTD–GβγCTD interface. (M) Color key for domains in p110γ, p101, and Gβγ.

Extended Data Fig. 5 Map quality of regions in Gβγ-bound PI3Kγ structures.

(A)-(C) Local resolution estimation for PI3Kγ·ADP–GβγHD, as well as states 1 and 2 of PI3Kγ·ADP–GβγHD–GβγCTD. (D)-(F) Visualization of sidechain density of residues in the p110γ subunit of PI3Kγ·ADP–GβγHD, and states 1 and 2 of PI3Kγ·ADP–GβγHD–GβγCTD. p110γ-C2-Tyr389 and p110γ-HD-Trp598 are labeled. (G)-(I) Overlay of maps and models showing the interfaces between p110γ-HD and -K domains in PI3Kγ·ADP–GβγHD, and states 1 and 2 of PI3Kγ·ADP–GβγHD–GβγCTD. Leu564 is indicated. (J)-(L) Overlay of maps and models showing the interfaces between the p101-CD and -CTD domains in PI3Kγ·ADP–GβγHD, and states 1 and 2 of PI3Kγ·ADP–GβγHD–GβγCTD. p101-Ile689 is indicated.

Extended Data Fig. 6 Structure and sequence alignment of PI3K isoforms at the GβγHD binding site of PI3Kγ.

Only PI3Kβ retains a hydrophobic signature that may be responsive to Gβγ. (A) Hydrophobic interactions between GβγHD and p110γ-HD. (B-D) Residues of PI3Kβ (PDB entry 2Y3A), PI3Kα (PDB entry 8DCP), and PI3Kδ (PDB entry 6ZAC) at positions corresponding to p110γ-Leu551 and -Leu574 are shown as stick models. (E) Sequence alignment of vertebrate p110-γ (PIK3CG), -β (PIK3CB), -α (PIK3CA), and -δ (PIK3CD) isoforms. Positions of interfacial residues Leu551, Leu564, and Leu574 from pig p110γ are indicated above the alignment.

Extended Data Fig. 7 Comparison of interfaces of p110γ-HD with GβγHD and p101-CTD with GβγCTD with those predicted by AlphaFold2.

(A) AlphaFold2 predicted residues on GβγHD (left panel) that interact with p110γ-HD (right panel). (B) AlphaFold2 predicted residues on GβγCTD (left panel) that interact with p101-CTD (right panel). (C) Experimentally determined interface residues on GβγHD (left panel) for interaction with p110γ-HD (right panel) in PI3Kγ·ADP–GβγHD. (D) Residues on GβγHD (left panel) involved in interaction with p110γ-HD (right panel) in state 1 of PI3Kγ·ADP–GβγHD–GβγCTD. (E) Residues on GβγCTD (left panel) that interact with p101-CTD (right panel) in PI3Kγ·ADP–GβγHD–GβγCTD. (F) Residues on GβγHD (left panel) that interact with p110γ-HD (right panel) in state 2 of PI3Kγ·ADP–GβγHD–GβγCTD. (G) Residues on GβγCTD (left panel) that interact with p101-CTD (right panel) in state 2 of PI3Kγ·ADP–GβγHD–GβγCTD.

Extended Data Fig. 8 Workflow of cryo-EM data processing of PI3Kγ.

(A) Representative micrograph (scale bar = 100 nm). About 5000 micrographs with similar outcomes were collected. (B) Representative 2D class averages (box size = 320 pixels where 1 pixel = 1.08 Å). (C) FSC plot, estimating 3.0 Å resolution using FSC at 0.143. (D) Cryo-EM reconstruction. In total, 269,357 particles were used for 3D map reconstruction. Map regions belonging to p110γ and p101 are roughly indicated by the black and red brackets. The CTD of p101 is colored magenta. (E) Local resolution estimation of the PI3Kγ cryo-EM map from (D). The overall resolution in most regions of p110γ is 2.5-3 Å with well-defined side chains, whereas the overall resolution of p101 is >3.5 Å. (F) Local refinement of PI3Kγ focused on the p101 NTD. The yellow circle indicates the fulcrum point used for local refinement. (G) Local refinement of PI3Kγ focused on the p101 C-terminal domain. The yellow circle indicates the fulcrum point used for local refinement. (H) Composite map generated from the three maps shown in panels (D), (F), and (G). (I) Local resolution estimation of the composite PI3Kγ cryo-EM map from (H). The overall resolution in most regions is 2.5-3 Å, whereas the p101 C-terminal domain has an improved resolution of 3-4 Å.

Extended Data Fig. 9 Workflow of cryo-EM data processing of PI3Kγ·ATP derived from an un-tilted dataset of PI3Kγ supplemented with Gβγ and ATP.

We collected 3936 frames for the PI3Kγ sample supplemented with Gβγ and ATP (white scale bar = 100 nm in the micrograph presented in the top left panel) and processed the data in CryoSPARC, resulting in 472,023 particles after several rounds of 2D classification. The 2D classes showing distinct Gβγ density are indicated by red boxes. The 2D classes showing no Gβγ density are indicated by yellow boxes. The classes in green boxes are undetermined. We generated 2 ab initio maps using the particles from the “red” and “green” 2D classes, followed by heterogeneous refinement in CryoSPARC, resulting in a “junk particle” and “Gβγ-bound PI3Kγ” cryo-EM maps. In parallel, we processed the particles from the “yellow” and “green” 2D classes using the same strategy, resulting in a “PI3Kγ alone” and another “junk particle” cryo-EM map. We then used the 3 maps (1: junk-particle, 2: PI3Kγ–Gβγ, and 3: PI3Kγ alone) for sorting all particles using heterogeneous refinement, and then did homogeneous and local refinements for the “PI3Kγ alone” data, resulting in a 3.3 Å cryo-EM reconstruction of PI3Kγ·ATP (Extended Data Fig. 2E).

Extended Data Fig. 10 Cryo-EM reconstruction of PI3Kγ·ATP.

(A) Cryo-EM map of PI3Kγ·ATP. Domains are colored as in Fig. 1a. (B) Superimposition of PI3Kα·diC4-PIP2 (PDB entry 4OVV, in yellow) with PI3Kγ·ATP (in black) indicates a steric clash between the PIP2 substrate and α36. diC4-PIP2 and ATP in PI3Kγ·ATP are shown as green and red spheres, respectively. The p110γ-RBD, K-N, and K-C domains are located in Areas 1, 2, and 3.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Tracking movies of migrating neutrophils in the head mesenchyme with p101 knockdown. The video shows the motility of neutrophils in 3 dpf Tg(lyzC:RFP) zebrafish larvae injected with buffer or p101 morpholino. Videos were recorded for 30 min at 1 min intervals. Representative videos from n = 3 independent experiments with four fish in each group are shown. Scale bar, 100 μm.

Supplementary Video 2

Tracking movies of migrating neutrophils in the head mesenchyme re-expressing p101 WT or mutations in the p101 knockdown background. The videos show the motility of neutrophils in 3 dpf Tg(lyzC:RFP) zebrafish larvae injected with buffer or p101 morpholino, then injected with Tol2-lyzC-human PIK3R5, mutant human PIK3R5 CTD-GS (p101CTD-GS) or PIK3R5 I692T (p101I692T) plasmids for transient re-expression. Videos were recorded for 30 min with 1 min intervals. Yellow tracks indicate neutrophils with transient p101 expression. Blue tracks indicate neutrophils with p101 knockdown in the same fish. Representative videos from n = 3 independent experiments with four fish in each group are shown. Scale bar, 100 μm.

Supplementary Video 3

3D flexible refinement of PI3Kγ·ADP–GβγHD–GβγCTD (top view). The flexible refinement of PI3Kγ·ADP–GβγHD–GβγCTD (combining state 1 and 2 particles) was carried out using the CryoSPARC 3D flexible refinement package. The regions corresponding to GβγHD and GβγCTD are indicated.

Supplementary Video 4

Supplementary Movie 4. 3D flexible refinement of PI3Kγ·ADP–GβγHD–GβγCTD (bottom view). The flexible refinement of PI3Kγ·ADP–GβγHD–GβγCTD (combining state 1 and 2 particles) was performed using the CryoSPARC 3D flexible refinement package. The regions corresponding to GβγHD and GβγCTD are labeled. The domains, including p110γ-KD, p110γ-HD, p110γ-C2 and p101-CTD, are indicated by arrows.

Supplementary Data 1

Uncropped gels for Supplementary Figs. 1a and 4a–f.

Supplementary Data 2

Source data for Fig. 4g,h.

Supplementary Data 3

RMSD and CCC from MDFF fitting.

Source data

Source Data Fig. 5

Source data for Extended Data Fig. 5c–j.

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Chen, CL., Syahirah, R., Ravala, S.K. et al. Molecular basis for Gβγ-mediated activation of phosphoinositide 3-kinase γ. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01265-y

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  • DOI: https://doi.org/10.1038/s41594-024-01265-y

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