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Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I

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Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I Lindsey N. Younga,b,c,1, Felix Goerdelera,2, and James H. Hurleya,b,c,3 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; bCalifornia Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; and cMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved September 18, 2019 (received for review July 6, 2019) Autophagy induction by starvation and stress involves the enzy- mechanisms of the many PI3KC3 regulators acting at the base of matic activation of the class III phosphatidylinositol (PI) 3-kinase the V have been undefined. complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is main- Nuclear Receptor Binding Factor 2 (NRBF2) is a positive reg- tained by inhibitory contacts between the VPS15 protein kinase ulator of PI3KC3-C1 that also acts at the base of the V shape (17). and VPS34 lipid kinase domains that restrict the conformation of the NRBF2 was identified as a PI3KC3-C1–associated factor through VPS34 activation loop. Here, the proautophagic MIT domain-containing proteomics of the mammalian autophagy network (18). Depletion protein NRBF2 was used to map the structural changes leading to of NRBF2 reduced autophagosome formation, implicating NRBF2 activation. Cryoelectron microscopy was used to visualize a 2-step as proautophagic (18). Work in Saccharomyces cerevisiae identified PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Atg38 as the homolog of NRBF2 and confirmed Atg38 to be a Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and proautophagic component of PI3KC3-C1 (19). The proautophagic releases the VPS34 kinase domain from the inhibited conformation. function of NRBF2 has been confirmed by most reports in mam- Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an malian cells (20–24), although contrary findings were reported by active conformation that has an unrestricted activation loop and is one group (24). NRBF2 and its ortholog Atg38 contain an N- poised for access to membranes. terminal Microtubule Interacting and Trafficking (MIT) domain that binds to PI3KC3-C1, followed by a central dimeric coiled-coil autophagy | cryo-EM | lipid kinase domain (17, 19, 25) (Fig. 1B). Dimerization can be decoupled from activation, as the N-terminal MIT domain (NRBF2MIT) alone is utophagy is a core cellular process, conserved throughout sufficient to enhance the kinase activity of the autophagy-specific Aeukaryotes, which is the central recycling system for the PI3-Kinase in vitro (17) and to rescue autophagy induction in removal of misfolded proteins, damaged organelles, and the MEFs (21). Here, we used cryoelectron microscopy (cryo-EM) and recycling of nutrients in starvation. Autophagic dysfunction is implicated in many disease states, including neurodegeneration, Significance immune disorders, cancer, and aging, among others (1). The class III phosphatidylinositol-3 kinase complexes (PI3KC3) I and II Autophagy is a prosurvival pathway of cellular self-consumption (PI3KC3-C1 and -C2), respectively, are essential for the initiation that is essential for the homeostasis of eukaryotic cells. Under- and expansion of autophagosomes (2–5). PI3KC3 generates the standing the switching of autophagy on and off at the struc- lipid phosphatidylinositol-3-phosphate, PI(3)P, which is recognized tural level has been a major goal in autophagy research. The by the WIPI proteins. WIPIs, in turn, recruit the machinery that active state structure is poorly stable and has until now not conjugates the autophagosomal marker LC3 to the expanding been possible to crystallize or to reconstruct by cryoelectron autophagosomal membrane (6). PI3KC3-C1 has been proposed to microscopy. We now provide a structure of the autophagy- be a promising therapeutic target for autophagy activators (7) be- initiating phosphatidylinositol 3-kinase (PI3K) lipid kinase com- cause the generation of PI(3)P is absolutely required for the re- plex I in the activated state. The structure shows how rear- cruitment of downstream autophagy proteins. There is considerable rangements in the right arm of the complex triggered by NRBF2 medical interest in selectively activating this pathway to promote lead the lipid kinase domain to move to a geometry with an human health and treat disease, yet there are no FDA-approved unrestricted activation loop and an active site poised to phos- pharmaceuticals that uniquely activate autophagy. phorylate membrane-embedded phosphatidylinositol (PI). PI3KC3-C1 consists of the lipid kinase VPS34, the putative serine/threonine protein kinase VPS15, the regulatory subunit Author contributions: L.N.Y. and J.H.H. designed research; L.N.Y. and F.G. performed research; L.N.Y. and J.H.H. analyzed data; and L.N.Y. and J.H.H. wrote the paper. BECN1, and the early autophagy-specific targeting subunit ATG14 Competing interest statement: J.H.H. is a scientific founder of, and receives research (8, 9). In PI3KC3-C2, ATG14 is replaced with UVRAG (10), while funding from, Casma Therapeutics. the other 3 subunits are preserved. The overall architecture of both This article is a PNAS Direct Submission. PI3KC3-C1 and -C2 has the shape of the letter V (11, 12) (Fig. Published under the PNAS license. 1A). The long coiled coils of BECN1 and ATG14 scaffold the left Data deposition: The EM density maps have been deposited in the Electron Microscopy arm in the standard view, with the membrane binding BARA do- Data Bank (EMDB), https://www.ebi.ac.uk/pdbe/emdb (accession no. EMDB-20387, MIT- main of BECN1 located at the outermost tip of the arm. PI3KC3- Fusion cryoEM map, mask for refinement, and FSC curve; accession no. EMDB-20390, C2 is inhibited by Rubicon and the HIV-1 protein Nef (13–15), activated PI3KC3-C1 and full-length NRBF2 cryo-EM map, mask used for refinement, and FSC curve). which regulate membrane docking by the tip of the left arm. The 1Present address: Division of Biological Sciences, University of California San Diego, La catalytic domains of the kinases VPS34 and VPS15 are at the tip of Jolla, CA 92161. the right arm (11, 12). PI3KC3 complexes are phosphoregulated by 2Present address: Department of Biochemistry, Freie Universität Berlin, 14195 Berlin, the Unc-51 like autophagy-activating kinase 1 (ULK1) complex Germany. (16), among other kinases (3). PI3KC3-C1 is inhibited by anti- 3To whom correspondence may be addressed. Email: [email protected]. apoptotic proteins Bcl-2/Bcl-XL (10). The binding of Bcl-2/Bcl- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. XL and the best-characterized phosphoregulatory modulations of 1073/pnas.1911612116/-/DCSupplemental. PI3KC3-C1 map to the base of the V. Thus far, the structural First published October 7, 2019. 21508–21513 | PNAS | October 22, 2019 | vol. 116 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1911612116 Downloaded by guest on September 29, 2021 complex (SI Appendix,Fig.S1D–G). Peptides within the N-terminal A PI3KC3-C1 B VPS15 domains of BECN1 and ATG14, the VPS15 helical solenoid, and BARA NRBF2 BARA kinase MIT coiled-coil the VPS34 C2 domain showed HDX decreases of up to 50%. BECN1 VPS34 WD40 kinase However, most HDX decreases throughout the remainder of the coiled-coil MIT domain ∼ – C2 solenoid 4 subunits did not exceed 10%. In the NRBF2 BECN1 fusion ATG14 complex, the regions that showed the highest protection were coiled-coil coiled-coil BH3 typically even more protected (SI Appendix, Fig. S1 D–G). Two C D regions of ATG14 that were ∼15% protected in the NRBF2 PI3KC3-C1 MIT-Fusion BECN1 BECN1 ATG14 VPS15 complex were ∼30% protected in the fusion, for example (SI ATG14 VPS15 VPS34 Appendix,Fig.S1F). Therefore, the presence of the fused MIT MIT NRBF2 generally recapitulates the qualitative protection domain pattern seen in the noncovalent complex with intact NRBF2, but the degree of protection is greater. This is presumably because the fused complex cannot dissociate when it is diluted into D2O for exchange experiments. Outside of the regions expected to E MIT-Fusion BARA interact with NRBF2, increased protection was also noted. For example, most of the catalytic domains of VPS34 and VPS15 WD40 VPS15 KINASE WD40 manifested a ∼20% increase in protection in the fusion (SI Ap- BARA pendix D G VPS34 VPS34 , Fig. S1 and ). These increases are suggestive of a C2 C2 more global decrease in protein dynamics in the fused construct. 180˚ BECN1 – – ATG14 Cryo-EM Structure of the BECN1 MIT Fusion Complex. Previous EM analyses of PI3KC3s have been limited by the dynamic character of solenoid BECN1 MIT ATG14 these complexes and by their tendency to be in preferred orien- tations on EM grids (11, 17, 25, 26). Even when preferred orien- MIT domain tations can be reduced or eliminated, the dynamics of the catalytic right arm of the complex (27) has still limited analysis (14). The MIT F GH apparent reduction in dynamics in the BECN1-NRBF2 form of BIOCHEMISTRY 180˚ PI3KC3-C1 suggested that this sample might be more tractable to cryo-EM than the WT PI3KC3-C1 and -C2 complexes. Two- MIT VPS34 kinase dimensional class averages of BECN1-NRBF2 PI3KC3-C1 showed additional features compared to WT PI3KC3-C1 (Fig. 1 C and D). A reconstruction was obtained with an overall resolution of 7.7 Å and local resolution ranging from 6.7 to 24 Å (Fig. 1 E and “ ” MIT Fig. 1. Cryo-EM structure of MIT-Fusion (BECN1-NRBF2 PI3KC3-C1). (A) F and SI Appendix, Fig. S2 and Table S1), showing the expected V- Schematic of PI3KC3-C1 containing VPS34, VPS15, BECN1, and ATG14.
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