The inhibitory nanobody NB7 binds tightly to all p110γ complexes and inhibits kinase activity, but does not prevent membrane binding

A. Cartoon schematic depicting nanobody inhibition of activation by lipidated Gβγ (1.5 µM final concentration). Lipid kinase assays show a potent inhibition of lipid kinase activity with increasing concentrations of NB7 (3-3000 nM) for the different complexes. The protein concentration of p110γ (300 nM), p110γ-p84 (330 nM) and p110γ-p101 (12 nM) was different due to intrinsic differences of each complex to be activated by lipidated Gβγ.

B. Association and dissociation curves for the dose response of His-NB7 binding to p110γ, p110γ-p84 and p110γ-p101 (50 – 1.9 nM) is shown. A cartoon schematic of BLI analysis of the binding of immobilized His-NB7 to p110γ is shown on the left. Dissociation constants (KD) were calculated based on a global fit to a 1:1 model for the top three concentrations and averaged with error shown.

B. Association and dissociation curves for His-NB7 binding to p110γ, p110α-p85α, p110β-p85β, and p110δ-p85β. Experiments were performed in duplicate with a final concentration of 50 nM of each class I PI3K complex.

D. Total Internal Reflection Fluorescence Microscopy (TIRF-M) analysis of the effect of nanobody NB7 on PI3K recruitment to supported lipid bilayers containing H-Ras(GTP) and farnesyl-Gβγ. Y647-p84/p110γ displays rapid equilibration kinetics and is insensitive to the addition of 500 nM nanobody (black arrow, 250 sec) on supported lipid bilayers containing H-Ras(GTP) and farnesyl-Gβγ.

E. Kinetics of 50 nM DY647-p84/p110γ membrane recruitment appears indistinguishable in the absence and presence of nanobody. Prior to sample injection, DY647-p84/p110γ was incubated for 10 minutes with 500 nM nanobody.

F. Representative TIRF-M images showing the localization of 50 nM DY647-p84/p110γ visualized in the absence or presence of 500 nM nanobody (+NB7). Membrane composition for panels C-E: 93% DOPC, 5% DOPS, 2% MCC-PE, Ras(GTP) covalently attached to MCC-PE, and 200 nM farnesyl-Gβγ.

Structure of p110γ bound to inhibitory nanobody NB7

A. Domain schematics of p110γ with helical domain (blue), activation loop (orange), and regulatory motif (green) of p110 annotated.

B. Density map of the p110γ-NB7 complex colored according to the schematic in A.

C. Cartoon model of the structure of p110γ bound to NB7 colored according to A.

D. Schematic depicting the key features of p110 and the nanobody binding site, colored according to panel A.

E. Domain schematic of NB7 CDR regions and their sequences.

F. Zoom in on the binding interface of NB7, with the CDRs colored as in panel E, and the electron density of the CDR regions contoured at 3σ (blue mesh).

PKC leads to dual phosphorylation of internal sites in the helical domain, with selectivity for apo p110γ and p110γ-p84 over p110γ-p101.

A. Putative phosphorylation sites mapped on the structure of p110γ (PDB: 7MEZ) and cartoon schematic. The regions are colored based on domain schematics featured in Fig 2A.

B. Raw MS spectra of the unphosphorylated and phosphorylated peptide for a region spanning 579-592 (RYESLKHPKAYPKL) and 593-607 (FSSVKWGQQEIVAKT).

C-E. Extracted traces and ratios of the intensity of extracted ion traces of different phosphorylation site peptides (Top to bottom: S594/S595 and S582) from © p110γ, (D) p110γ/p84 or (E) p110γ/p101 samples treated with increasing concentration of PKCb according to the legend.

Activating phosphorylation at the helical domain leads to opening of the regulatory motif

A. HDX-MS comparing apo and phosphorylated p110γ. Significant differences in deuterium exchange are mapped on to the structure and cartoon of p110γ according to the legend (PDB: 7MEZ).

B. The graph of the #D difference in deuterium incorporation for p110γ, with each point representing a single peptide. Peptides colored in red are those that had a significant change in the mutants (greater than 0.4 Da and 5% difference at any timepoint, with a two tailed t-test p<0.01). Error bars are S.D. (n=3).

C. Representative bimodal distribution (EX1 kinetics) observed in the helical domain peptides of p110γ.

D. Representative p100γ peptides displaying increases in exchange in the phosphorylated state are shown. For all panels, error bars show SD (n = 3)

E. Lipid kinase activity assays of phosphorylated and non-phosphorylated p100γ (concentration, 12nM to 1000nM) ATPase activity (left) and membrane (5% phosphatidylinositol 4,5-bisphosphate (PIP2), 95% phosphatidylserine (PS)) activation (right). Significance is indicated by **(<0.001%), and ***(<0.0001%).

Nanobody NB7 blocks PKC phosphorylation, and phosphorylation prevents nanobody binding

A. Graph showing the intensities of phosphorylated and non-phosphorylated p110γ peptide (593-607) for PKC (500 nM) treated p110γ (black), PKC treated p110γ with NB7 (red) and PKC treated p110γp101 (purple). Scatter plot showing the percent phosphorylation of each complex from the left graph for the indicated peptide (n=3, right). Significance is indicated by ***(<0.0001%).

B. Graph showing the intensities of phosphorylated and non-phosphorylated p110γ peptide (579-592) for PKC treated p110γ (black), PKC treated p110γ with NB7 (red) and PKC treated p110γp101 (purple). Scatter plot showing the percent phosphorylation of each complex from the left graph for the indicated peptide (n=3, right). Significance is indicated by * (<0.01%), and ***(<0.0001%).

C. Cartoon schematic of BLI analysis of the binding of immobilized His-NB7 to phosphorylated and non-phosphorylated p110γ.

D. Association curves for phosphorylated and non-phosphorylated p110γ (25nM) binding to His-NB7 are shown (n=3).

E. Kinase activity assays comparing the activation/inhibition of phosphorylated and non-phosphorylated p110γ (concentration, 12nM to 1000nM) with or without nanobody (3000nM final). Significance is indicated by * (<0.05%), and NS (>0.05%).

Comparison of nanobody binding site compared to p85 inhibition of class IA PI3Ks and class IB activation sites

A. Comparison of the nanobody NB7binding site in p110γ compared to the nSH2 inhibitory site in p110((PDB: 3HHM) (Mandelker et al., 2009)

B. Comparison of the nanobody NB7binding site in p110γ compared to the X-ray structure of the Ras binding site (PDB: 1HE8) (Pacold et al., 2000) and the Alphafold model of Gβγ bound to p110γ (Rathinaswamy et al., 2023).

C. Oncogenic mutations and post-translational modifications in spatial proximity to the nanobody binding site.

p110γ-NB7 complex cryo-EM analysis workflow (related to main figure 2):

cryo-EM processing workflow of p110γ-NB7 complex are shown in order of a representative micrographs, representative 2D classification and 3D reconstruction processing strategy. Bottom left shows Gold-standard Fourier shell Correlation (FSC) curve of final round on non-uniform homogenous refinement.

Comparison of full length p110γ bound to NB7 compared to p110γ-p101 (related to main figure 2):

The structure of the p110γ-p101 complex (PDB:7MEZ) compared to the NB7-p110γ complex is shown colored according to B factor based on the legend.

(related to main figure 3)

MS/MS spectra of peptides spanning S582 and S594/S595 for both phosphorylated and unphosphorylated states. The theoretical and experimental mass are annotated for all peptides.

Cryo-EM data collection, refinement and validation statistics (related to main figure 2)

HDX-MS data collection and validation statistics (related to main figure 4)