Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation

Abstract

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

Fig. 1. Cryo-EM structure of the p110γ p101 complex.

(A) Domain schematic of Homo sapiens p110γ and Sus scrofa p101 used in this study. (B) Gel filtration elution profile of different p110γ complexes (i.e., apo or bound to p84, p101, and p101-NB1-PIK3R5). An SDS–polyacrylamide gel electrophoresis image of the p110γ-p101-NB1-PIK3R5 complex is shown, with molecular weight (MW) standards indicated. (C) Lipid kinase activity assays of different p110γ complexes (concentration, 30 to 3000 nM) with and without lipidated Gβγ (1.5 μM concentration) using 5% phosphatidylinositol 4,5-bisphosphate (PIP₂) vesicles mimicking the plasma membrane (20% phosphatidylserine, 50% phosphatidylethanolamine, 10% cholesterol, 10% phosphatidylcholine, 5% sphingomyelin, and 5% PIP₂). The fold change upon Gβγ activation is indicated. Every replicate is plotted, with error shown as SD (n = 3 to 6). Two-tailed P values represented by the symbols as follows: ** < 0.001; * < 0.02; N.S. > 0.02. (D) Density map of the p110γ-p101-NB1-PIK3R5 complex colored according to the schematic in (A). (E) Cartoon representation of the p110γ-p101 complex colored according to the schematic in (A). (F) Cartoon schematic of the p110γ-p101 complex.

Fig. 2. Structural basis of the p110γ-p101 binding interface.

(A) Cartoon representation of the p110γ-p101 complex, with p101 colored as in Fig. 1 and p110γ colored according to the attached schematic, with p101-interacting regions (RBD-C2 linker, C2, and the C2-helical linker) indicated. Important features are shown in a cartoon schematic. (B) Interaction between the GBD of p101 and the C2 domain of p110γ. The p110γ C2 domain is shown as an electrostatic surface with p101 shown as sticks. (C) The electrostatic surface of the GBD of p101. A cartoon schematic highlighting potential electrostatic interactions between the GBD of p101 and the C2 domain of p110γ and membranes. (D) The structure of p110γ-p101 complex highlighting the orientation of the p110γ ABD, with p101 shown as a transparent surface. The different domains are colored as indicated according to the cartoon schematic. (E) The ABD of p110γ coordinates the RBD-C2 linker of p110γ to interact with p101. The RBD-C2 linker, ABD, and the region of p101 that binds the RBD-C2 linker are shown in a cartoon representation. The electron densities of the ABD interface, RBD-C2 linker, and region of p101 that binds the RBD-C2 linker are visible.

Fig. 3. Class IA and IB PI3Ks form distinct interfaces with regulatory subunits and the ABD.

(A) The structure of p110γ-p101 complex, with p110γ shown as a surface, p101 shown as a ribbon, and the domains colored according to the cartoon schematic as indicated in (E). (B) The structure of p110α-p85α complex [Protein Data Bank (PDB): 4JPS], with p110α shown as a surface, the nSH2 and iSH2 domains of p85α shown as a ribbon, and the domains colored according to the cartoon schematic as indicated in (E). (C) The ABD of p110γ does not interact with either the regulatory subunit or kinase domain. The p110γ-p101 complex is shown as in (A). (D) The ABD of p110α interacts with both the regulatory subunit and the kinase domain. The p110α-p85α complex is shown as in (B). The altered orientation of the ABD compared to p110γ is indicated by the black arrow. (Cartoon schematics indicating the differences between class IA and class IB are shown for (A) to (D). (E) Domain schematic comparing the interactions between p110 catalytic and the p101/p85 regulatory subunits. Inhibitory interactions are colored in red, with interacting regions indicated by the arrows.

Fig. 4. Disease-linked and engineered p110γ mutations at the interface with p101 and the ABD modulate Gβγ activation.

(A) Somatic mutations found in PIK3CG from the COSMIC database are indicated on the structure, with frequency indicated by the legend. Mutations found in more than 7 tumors are shown as sticks, with mutations found in more than 11 tumors shown as spheres. The orientation of residues around mutations located at the p110γ-p101 interface (i, E347) and ABD interface (ii, R472) are shown. (B) Mutations do not disrupt the p110γ-p101 complex. Gel filtration elution profiles of complexes of p110γ (WT, E347, and R472) bound to p101. (C) Mutations at the p110γ-p101 and ABD interface can lead to enhanced activation by Gβγ. Lipid kinase activity assays of different p110γ complexes (concentration, 10 to 1000 nM) with and without Gβγ (concentration indicated). (D) HDX-MS revealed enhanced protein dynamics at p101 and ABD interfaces induced by E347K and R472 mutants. Peptides showing significant deuterium exchange differences (>5%, >0.4 kDa, and P < 0.01 in an unpaired two-tailed t test) between p110γ-p101 complexes of WT and E347K (left) and WT and R472C (right) are colored on a cartoon model of p110γ-p101 according to the legend. (E) The GBD is dynamic in solution but is stabilized by nanobody (NB1-PIK3R5) binding. Electron density maps of p110γ-p101 alone (left) and p110γ-p101 bound to NB1-PK3R5 (right). (F) Charged residues in p110γ-p101 mediate the interaction of the p110γ C2 domain to the p101 GBD. (G) Mutation of the p110γ-C2 p101-GBD interface (p110γ D369R) leads to decreased activation by Gβγ. Biochemical assays in (C) and (G) were carried out with p110γ-p101 complexes (concentration, 10 to 1000 nM) and Gβγ (concentration indicated). Five percent PIP₂ membranes were made mimicking the plasma membrane. Every replicate is plotted, with error shown as SD (n = 3 to 9). Two-tailed P values represented by the symbols in panels C+G as follows: ** < 0.001; * < 0.02.

Fig. 5. Full activation of p110γ by lipidated Gβγ requires the GBD domain of p101, and the GBD-Gβγ interface can be disrupted by a p101-specific nanobody.

(A) HDX-MS revealed that interaction of p110γ-p101 with membranes leads to altered protein dynamics in both the p110γ and p101 subunits, with stabilization of the GBD of p101. For (A) to (C), peptides showing significant deuterium exchange differences (>5%, >0.4 kDa, and P < 0.01 in an unpaired two-tailed t test) between conditions are colored on a cartoon model of p110γ-p101 according to the legend in (B). A cartoon schematic is shown indicating the two conditions compared using HDX-MS. (B) HDX-MS revealed that interaction of p110γ-p101 with lipidated Gβγ subunits stabilizes the GBD and C2-helical/helical domain of p110γ. The HDX-MS data from (A) and (B) are from our previous study (32) and have been mapped onto the p110γ-p101 structure. (C) HDX-MS revealed that interaction of p110γ-p101 with NB1-PIK3R5 protects the same surface of GBD that is stabilized upon binding Gβγ on membranes. (D) Biolayer interferometry analysis of the binding of the immobilized NB1-PIK3R5 nanobody to p110γ-p101. (E) The NB1-PIK3R5 nanobody specifically inhibits only the p110γ-p101 complex from GPCR activation while not affecting the p110γ-p84 complex. Biochemical assays were carried out with p110γ-p101 (50 to 3000 nM) and p110γ-p84 (1500 to 3000 nM) using plasma membrane mimic vesicles with and without NB1-PK3R5 (6 μM). Lipidated Gβγ was present at 1.5 μM concentration. (F) IC₅₀ measurement of p110γ-p101 inhibition using varying concentrations of the NB1-PIK3R5 nanobody in the presence of 600 nM Gβγ. For (E) and (F), every replicate is plotted, with error shown as SD (n = 3 to 6). Two-tailed P values represented by the symbols in panel E as follows: ** < 0.001; * < 0.02; N.S. > 0.02. (G) Model of the inhibition of GPCR activation of the p110γ-p101 complex by the NB1-PIK3R5 nanobody.

Fig. 6. Single-molecule characterization of p110γ-p101 reveals that both subunits can engage membrane-anchored Gβγ.

(A) Schematic showing proteins examined using the single-molecule fluorescence approach. Experiments measured the association of fluorescently tagged proteins (Alexa Fluor 488–SNAP–Gβγ, DY647-p110γ, and DY647-p101-p110γ) to an SLB. DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-l-serine. (B) Membrane association of DY647-p110γ or DY647-p101-p110γ requires membrane-anchored Gβγ. Single-molecule localization measurements were measured in the presence of either 100 pM DY647-p110γ or 10 pM DY647-p101-p110γ. The small number of DY647-p110γ (+Gβγ) seen in the snap shot image reflects the transient nature of the interaction between DY647-p110γ and Gβγ. (C and D) Single-molecule dwell time distributions of DY647-p110γ or DY647-p101-p110γ, measured in the presence of membrane-anchored Gβγ. DY647-p110γ transiently associates with membrane-anchored Gβγ (C, τ₁ = 22 ms, n = 2832 events). DY647-p101-p110γ binds strongly to membrane-anchored Gβγ [τ₁ = 0.334 s (31%), τ₂ = 1.31 s (69%), n = 3996 events]. (E) Gβγ membrane density–dependent changes in the membrane binding behavior of DY647-p101-p110γ. Concentration of Gβγ represents the solution concentration. (F) DY647-p101-p110γ absorption kinetics at different Gβγ membrane densities. (G) Model of p110γ-p101 recruitment to Gβγ subunits at both low and high membrane densities.

Fig. 7. Model for regulation of p110γ-p101 activation by Gβγ membrane density and modulation by nanobodies and disease-linked mutations.

(A) Schematic of how Gβγ subunits can lead to p110γ-p101 activation at different Gβγ surface densities and how this can be disrupted by the NB1-PIK3R5 nanobody. (B) Schematic of how mutations at the p101 and ABD interfaces in p110γ can lead to enhanced Gβγ activation and how disruption of the GBD-C2 interface can lead to decreased Gβγ activation.