Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I

Abstract

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

Keywords: autophagy; cryo-EM; lipid kinase.

Fig. 1.

Cryo-EM structure of “MIT-Fusion” (BECN1-NRBF2^MIT PI3KC3-C1). (A) Schematic of PI3KC3-C1 containing VPS34, VPS15, BECN1, and ATG14. (B) Schematic of NRBF2. (C) Two-dimensional cryo-EM class average of PI3KC3-C1 for comparison, illustrating the mobility of the VPS34 kinase domain. (D) Two-dimensional cryo-EM class average of MIT-Fusion. (E) Cryo-EM reconstruction of MIT-Fusion with VPS34 helical and catalytic domains masked out. Models originate PDB depositions for yeast PI3KC3-C2 (5DFZ), NRBF2^MIT (4ZEY), and BECN1^BARA (4DDP). The cryo-EM map has been deposited in the EMDB under accession no. EMDB-20387 (34). (F) Local resolution of MIT-Fusion structure with VPS34 helical and kinase domains included in masking, shown at a contour level of 10 σ or 0.0097 in Chimera and rotated 180° in G. (H) The VPS34 catalytic domain is visible (arrow), albeit at a low resolution of 18–24 Å, when contoured at a threshold of 6 σ or 0.0058 in Chimera.

Fig. 2.

Two copies NRBF2^MIT are required for activation. (A) Activity assay of PI3KC3-C1 and MIT-Fusion, with or without NRBF2^MIT or NRBF2. The average of 3 experiments performed in duplicate and normalized to PI3KC3-C1 activity are shown. The error bars represent the SEM, and P values were determined between PI3KC3-C1 and NRBF2^MIT or NRBF2, and MIT-Fusion and NRBF2. (B) Negative stain 2D class average of MIT-Fusion and MIT-Fusion incubated with MBP- NRBF2^MIT. (C) Two-dimensional cryo-EM class average of full-length NRBF2 bound to PI3KC3-C1.

Fig. 3.

CryoEM structure containing PI3KC3-C1 and full-length, dimeric NRBF2. (A) Cryo-EM reconstruction of a sample containing PI3KC3-C1 and full-length NRBF2 (PI3KC3-C1:2NRBF2) contoured at 10 σ or 0.0144 in Chimera. The cryo-EM map has been deposited in the EMDB under accession no. EMDB-20390 (35). Models docked into cryo-EM reconstruction include yeast PI3KC3-C2 (5DFZ), VPS34 kinase domain (4PH4), NRBF2^MIT (4ZEY), and BECN1^BARA (4DDP). (B) Close-up of the cryo-EM reconstruction of the VPS15 kinase (cyan, PDB ID code 5DFZ) and VPS34 kinase domains (blue, PDB ID code 4PH4), activation loop of VPS34 in red. (C) Close-up of the cryoEM reconstruction for the regions contacting NRBF2^MIT (red) include density for a helix, putatively, the BH3 helix of BECN1 (orange). (D) Close-up of solenoid of VPS15 (cyan) ,which provides an extensive face for MIT binding. (E) Cartoon representation of α-helices-1, 2, 3 of NRBF2^MIT contacting α-helices 12, 14, 16 of the VPS15 solenoid as well as the putative BH3 helix. (F) Local resolution estimates for PI3KC3:2NRBF2, with the VPS34 catalytic domain defined at 8–18 Å resolution (arrow).

Fig. 4.

Activation mechanism for transitioning PI3KC3-C1 from its inhibited to its activated state. (A) Top-down view of the inhibited PI3KC3-C1 (yeast PI3KC3-C2, 5DFZ) state, the VPS34 kinase domain sits perpendicular to VPS15. Top-down view of the activated PI3KC3-C1 (models from PDB ID codes 5DFZ, 4PH4, 4ZEY, 4DDP) state docked into the cryo-EM reconstruction (EMDB-20390) (35), the VPS15 kinase domain shifts by 10 Å to come more in line with rest of the complex. While VPS34 pivots 25°, the base of the VPS34 kinase domain moves by 5 Å while the top moves by 45 Å. (B) The inhibited state (pink) of VPS15 moves 10 Å away from VPS34 (blue) to reach its activated state (cyan), exposing the activation loop (red) of VPS34. (C) In the inhibited state, the activation loop (red) of VPS34 (lavender, from inhibited state 5DFZ) is occluded by the VPS15 kinase domain (pink, PDB ID code 5DFZ). (D) In the activated state, the activation loop (red) of VPS34 (blue) is released from VPS15 (cyan) inhibition and is more available to engage substrate. (E) Activation model of PI3KC3-C1 on membranes, in the inhibited state, the activation loop of VPS34 is not in a position to engage substrate. In solution, PI3KC3-C1 is highly dynamic, and the VPS34 kinase domain can dislodge (light pink) from the complex to directly bind membranes without the benefit of the rest of the complex. In the activated state, VPS34 is positioned in a precise geometry such that the activation loop of VPS34 is accessible to substrate in the membrane.