Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

Abstract

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.

Keywords: mass spectrometry; mutants; phosphoinositide 3-kinase (PI3K).

Fig. 1.

Cryo-EM structure of the PI3Kα-H1047R complex. (A) Location of H1047 mutation within the functional domains of PI3Kα. The domains are color-coded, and the linker sequences between the domains are shown in gray in the models and electron density maps. (B) SDS-PAGE (left) and native gel (right) images of the purified PI3Kα-H1047R complex; MW, molecular weight. (C) Kinase activity of the PI3Kα-H1047R complex measured by an HTRF assay. The star (*) indicates the presence of 150 nM BYL-719 during protein expression. Data shown represent mean ± SEM of three independent measurements conducted in triplicate. (D and E) Cryo-EM density map (D) and corresponding model (E) for the PI3Kα-H1047R complex bound to BYL-719 (orange). (F) The rmsd of Cα atoms between the cryo-EM models of the BYL-719–bound PI3Kα-H1047R and WT PI3Kα (PDB ID: 7MYO). (G) Cα rmsd between the cryo-EM model and the crystal structure model for the inhibitor-bound PI3Kα-H1047R (PDB ID: 3HHM).

Fig. 2.

Structural comparison between the PI3Kα-H1047R and WT PI3Kα complexes. (A–C) Unique conformation of R1047 in the cryo-EM structure of the PI3Kα-H1047R complex (A) that is distinct from the previously reported inward (B) or outward (C) conformations of H/R1047. (D) BYL-719-binding mode comparison between the cryo-EM structures of the PI3Kα-H1047R and WT PI3Kα (PDB ID:7MYO) complexes. (E) Structural comparison of the ABD of p110α and iSH2 domain of p85α between the cryo-EM structures of PI3Kα-H1047R and WT PI3Kα (PDB ID: 7MYO).

Fig. 3.

Cryo-EM structure of the PI3Kα-E542K complex. (A) SDS-PAGE (left) and native gel (right) images of the purified PI3Kα-E542K complex. (B) Kinase activity of the PI3Kα-E542K complex measured by an HTRF assay. The star (*) indicates the presence of 150 nM BYL-719 during protein expression. Data shown are mean ± SEM of three independent measurements conducted in triplicate. (C) Cryo-EM density map (top) and corresponding structural model (bottom) of the PI3Kα-E542K complex bound to BYL-719 (orange). (D and E) Structural comparison of the residues around E/K542 (D) or the ATP binding site (E) among the cryo-EM structures of the PI3Kα-E542K and WT PI3Kα (PDB ID: 7MYO) complexes and the crystal structure of p110α without an ABD (ΔABD p110α; PDB ID: 6OAC). The nSH2 domain of p85α in WT PI3Kα (PDB ID: 7MYO) is shown in gray (E).

Fig. 4.

Cryo-EM structure of the PI3Kα-E545K complex. (A) SDS-PAGE (left) and native gel (right) images of the purified PI3Kα-E545K complex. (B) Kinase activity of the PI3Kα-E545K complex measured by an HTRF assay. The star (*) indicates the presence of 150 nM BYL-719 during protein expression. Data shown are mean ± SEM of three independent measurements conducted in triplicate. (C) Cryo-EM density maps (top) and corresponding models (bottom) for the PI3Kα-E545K bound to BYL-719 (orange) shown in two orientations. (D) Cα rmsd between the cryo-EM models of PI3Kα-E545K and PI3Kα-E542K. (E) Structural comparison of the residues around E/K545 between the cryo-EM structures of PI3Kα-E545K and WT PI3Kα (PDB ID: 7MYO) complexes.

Fig. 5.

Confidence map analysis of the PI3Kα mutants. (A) Comparison of ED in the confidence maps of PI3Kα-H1047R (left) and WT PI3Kα (right; PDB ID: 7MYO) at 1% FDR. (B and C) Two regions of ED (salmon) are observed in the confidence maps of PI3Kα-E542K (B) and PI3Kα-E545K (C) at 1% FDR. (D and E) Mapping of these two regions of ED in the structure models of the PI3Kα-E542K (gray; D), PI3Kα-E545K (sky blue; E), and superimposed WT PI3Kα (plum; PDB ID: 7MYO). The ABD of p110α is highlighted in magenta, while p85α is omitted for clarity. The resolutions of these two regions are insufficient to model, but the positions are similar to the unmodeled ABD (ED-α) and the C-terminus (ED-β) of p110α.

Fig. 6.

Schematic models representing key structural features of phosphopeptide- and mutant-activated PI3Kα. (A) The inhibitor BYL-719 occupies the ATP binding site and stabilizes the binding of the cSH2, SH3, and BH domains to the catalytic core. The cSH2 domain is consistently positioned near the active site of p110α, blocking approach to the cell membrane and preventing access of ATP. BYL-719 thus acts as an allosteric inhibitor in addition to competing with ATP. (B) In apo PI3Kα, the SH3, BH, and cSH2 domains of p85α are more dynamic and do not consistently inhibit p110α. (C) The binding of phosphopeptide to the nSH2 and cSH2 domains of p85α triggers the disengagement of most p85α domains from p110α and induces mobility of the ABD, which remains bound to the iSH2 domain (not shown in the drawing). This structural transformation facilitates the interaction of p110α with the cell membrane. (D) The H1047R mutation reorganizes the local conformations in the C-terminal region of the kinase domain. It also reduces regulatory interactions between cSH2, SH3, BH, and the catalytic core. Further conformational changes are observed in the iSH2 domain and ABD. (E) The E542K and E545K mutations abolish the tight interaction between the helical domain of p110α and the nSH2 domain of p85, lead to the disengagement of most p85α domains from p110α, and mimic activation of PI3Kα by a phosphopeptide (48).