Cryo-EM structures of PI3Kα reveal conformational changes during inhibition and activation

Abstract

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.

Keywords: activation; activity-dependent conformational changes; inhibition; phosphoinositide 3-kinase (PI3K).

Fig. 1.

Cryo-EM structure of PI3Kα. (A) SDS-PAGE image of the purified PI3Kα complex. (B) Native gel image of the purified PI3Kα complex. (C) Selected 2D class averages of the PI3Kα complex. (D) Cryo-EM density maps and corresponding models of the PI3Kα complex in two orientations. (E) Confidence maps of cryo-EM, at 1% FDR, colored by local resolution. (F) Domain structure of p110α and p85α.

Fig. 2.

Differences between the cryo-EM structure (gold) and crystal structure (aqua) of PI3Kα. (A) Rmsd between cryo-EM and crystal structures (PDB ID code 4OVU) of PI3Kα. (B) The ABD and iSH2 domains are displaced by 6.9 Å at the Cα of S505. (C) The loops in the N-lobe of the kinase domain are displaced at both A775 and I778. (D) A flexible loop of the nSH2 domain is displaced by 11 Å as measured at the Cα of H365. (E) The nSH2 domain is rotated relative to the helical domain by 14°.

Fig. 3.

Cryo-EM structure of PI3Kα bound to BYL-719. (A) BYL-719 significantly reduces the kinase activity of PI3Kα as determined by membrane capture lipid kinase assay. (B) Selected 2D class averages of PI3Kα bound to BYL-719. (C) Cryo-EM density maps and corresponding models for PI3Kα bound to BYL-719 (gold) shown in two orientations. (D) Confidence maps of cryo-EM, at 1% FDR, colored by local resolution, showing a significant volume of low-resolution electron density. (E) Rmsd between the cryo-EM model and the crystal structure model for BYL-719–bound PI3Kα (PDB ID code 4JPS). (F) BYL-719 (yellow) bound to the active site of p110α forms hydrogen bonds with Q859 and S854 which drive p110α selectivity. The CF₃ group is in a different orientation and forms a hydrogen bond with S774.

Fig. 4.

Low-resolution extra density in PI3Kα structures. (A) Three regions of extra density are observed in PI3Kα and BYL-719–bound structures. (B) The extra density in BYL-719–bound PI3Kα. The resolution of these domains is insufficient to model, but the volumes are similar to the unmodeled SH3, BH, and cSH2 domains. We suggest that ED1 is the cSH2 domain (red), ED2 is the BH domain (green), and ED3 (orange) is the SH3 domain. (C) ED2 interacts with several amino acids on the surface of PI3Kα, including H1047R, the N- and C-lobes of the kinase domain, and the iSH2 domain. The extra density also interacts directly with BYL-719.

Fig. 5.

3DVA of unbound PI3Kα contains a DFG-out conformation. (A) The electron density map of the DFG motif of PI3Kα shows a DFG-in conformation. (B) 3DVA of PI3Kα shows that the DFG motif can also adopt a DFG-out conformation. This conformation is a minor component of the complete dataset.

Fig. 6.

Activation of PI3Kα with an activating phosphopeptide as observed by cryo-EM. (A) Quantification of lipid kinase activity from membrane capture assay upon stimulation with a phosphopeptide derived from PDGFRβ. (B) Native gel electrophoresis of PI3Kα with phosphopeptide. p110α and p85α dimerization is not disrupted by incubation with the PDGFRβ-derived phosphopeptide. (C) Comparison of a 2D class average of phosphopeptide-bound PI3Kα (Top Left) with an electron density map derived from a crystal structure of p110α (PDB ID code 6OAC) (Bottom Left) and 2D class averages of unbound and BYL-719–bound PI3Kα (Right) in similar orientations for comparison. (D) Models of the catalytic core (Top) and PI3Kα (Bottom) in the same orientation as (C) for comparison.