Structural pathway for class III PI 3-kinase activation by the myristoylated GTP-binding pseudokinase VPS15

The class III phosphatidylinositol (PI) 3-kinase complexes I and II (PI3KC3-C1 and -C2) are central to the initiation of macroautophagy and endosomal maturation, respectively. Through three-dimensional classification of a large cryo-EM dataset of human PI3KC3-C1 bound to the small GTPase RAB1A, we were able to map the structural pathway of enzyme activation. The inactive conformation is stabilized by an N-myristoyl modification of the pseudokinase (PK) subunit VPS15. The N-myristate is sequestered in the N-lobe of the VPS15 PK domain, which stabilizes a series of interactions whereby VPS15 sequesters and blocks the catalytic and membrane binding units of the VPS34 lipid kinase. In the activated conformation, the N-myristate and the VPS34 lipid kinase domain are liberated to interact with membranes and catalyze PI3P formation. The VPS15 PK domain contains a unique Arg at the gatekeeper position and binds tightly to GTP. GTP binding structurally stabilizes the N-myristate "in" conformation, which promotes the inactive conformation. This pathway provides a general mechanism for PI3KC3 activation in autophagy and endosome biogenesis and a roadmap for their pharmacological upregulation.