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.

Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes

“…We have previously shown that yeast complex II has a Y-shaped structure, with the one arm bearing Vps34 and Vps15 acting as the catalytic arm, while the other arm bearing Vps30 and Vps38 is the adaptor arm ( Rostislavleva et al, 2015 ). A similar structural organization has been observed for human complexes I and II ( Young et al, 2019 ; Baskaran et al, 2014 ; Ma et al, 2017 ). The aromatic finger motif in the BARA domain of Vps30 is important for membrane binding and kinase activity of complex II, shedding light on the importance of the adaptor arm for membrane binding and kinase activity.…”

The Role of Autophagy in Gastric Cancer Chemoresistance: Friend or Foe?

“…In addition, the N-terminal 640 residues (NTD) of FIP200, shaping like a letter C with the presence of ATG13, has an intimate interaction with C-terminal IDR of ATG13 and C-terminal early autophagy targeting/tethering (EAT) domain of ULK1, ensuring the successful initiation of autophagy ( Shi et al, 2020 ). Following the activation of ULK1 complex, the class III phosphatidylinositol 3-kinase (PI3KC3) complex, which consists of vacuolar protein sorting 34 (VPS34, also known as PI3KC3), ATG14, the activating molecule in BECN1-regulated autophagy protein 1 (AMBRA1), and the scaffold protein Beclin-1, generates phosphatidylinositol 3-phosphate (PI3P) at an endoplasmic reticulum (ER) subdomain named omegasome ( Young et al, 2019 ; Sanchez-Martin et al, 2020 ). Subsequently, PI3P recruits certain effector proteins, including WIPIs (WD repeat domain phosphoinositide-interacting proteins) ( Figure 2B ), which can bind ATG16L1 ( Bakula et al, 2017 ).…”

Structural Biology and Electron Microscopy of the Autophagy Molecular Machinery

“…One major difference is that the ATG14L CTD in the human PI3KC3-C1 complex contacts the membrane on one tip instead of Vps30 as proposed for the PI3KC3-C2 complex in yeast. Besides the intact complexes, structures of the human PI3KC3-C2 bound to the PI3KC3-C2 binding domain (PIKBD) of its endogenous inhibitor Rubicon [79] and the human PI3KC3-C1 bound to its positive regulator Nuclear Receptor Binding Factor 2 (NRBF2) [73] have also been recently determined by cryo-EM. Rubicon was found to bind to the BECN1 BARA domain, likely inhibiting the interaction of PI3KC3-C2 with the membrane.…”

“…Due to its transmembrane and intrinsically disordered nature, structural studies on ATG9 are notoriously challenging. Nevertheless, our group has successfully determined the structure of the trimeric Arabidopsis ATG9 by cryo-EM at 7.8 Å resolution, revealing the overall architecture and domain organization [73]. Although no atomic model is available for ATG9, we have generated the first putative model of the protein by integrating co-evolutionary information and homology modelling approaches.…”

“…Upon autophagy initiation, an isolated membrane known as the phagophore is formed at diverse membrane sites such as endoplasmic reticulum (ER) subdomains, mitochondria-ER contact sites, the ER-Golgi intermediate Despite extensive research over the past decades, our knowledge on the underlying molecular mechanisms of autophagosome biogenesis remains far from complete [76,77]. Recent studies using single-particle electron microscopy (EM) has contributed remarkable progress in the structural elucidation of several ATG proteins [46,67,73,78,79]. Here we focus on the structural biology aspect of the autophagosome biogenesis with emphasis on studies by single-particle EM and discussed the structure-function relationship of the core ATG proteins involved in autophagy initiation.…”