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Structural Basis for Autophagy Inhibition by the Human Rubicon–Rab7 Complex

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Structural Basis for Autophagy Inhibition by the Human Rubicon–Rab7 Complex Structural basis for autophagy inhibition by the human Rubicon–Rab7 complex Hersh K. Bhargavaa,b,1, Keisuke Tabatac, Jordan M. Bycka,b, Maho Hamasakid, Daniel P. Farrelle,f, Ivan Anishchenkoe,f, Frank DiMaioe,f, Young Jun Img, Tamotsu Yoshimoric,d, and James H. Hurleya,b,h,2 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; bCalifornia Institute for Quantitative Biosciences, University of California, Berkeley CA 94720; cDepartment of Genetics, Graduate School of Medicine, Osaka University, 565-0871 Osaka, Japan; dLaboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, 565-0871 Osaka, Japan; eDepartment of Biochemistry, University of Washington, Seattle, WA 98195; fInstitute for Protein Design, University of Washington, Seattle, WA 98195; gCollege of Pharmacy, Chonnam National University, Gwangju, 61186, Republic of Korea; and hMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by Brenda A. Schulman, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved June 3, 2020 (received for review April 24, 2020) Rubicon is a potent negative regulator of autophagy and a autophagy regulators, which also includes PLEKHM1 and Pacer (9, potential target for autophagy-inducing therapeutics. Rubicon- 17), two other proteins that modulate late steps in autophagy. The mediated inhibition of autophagy requires the interaction of the RH domains of Rubicon, PLEKHM1, and Pacer all contain nine C-terminal Rubicon homology (RH) domain of Rubicon with Rab7– conserved cysteines and one conserved histidine, which have been GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH predictedtobinddivalentzinccations and are required for Rubicon domain in complex with Rab7–GTP. Our structure reveals a fold for and PLEKHM1 to interact with Rab7 (9). Despite the importance the RH domain built around four zinc clusters. The switch regions of the RH domain in targeting Rubicon and other regulators of the of Rab7 insert into pockets on the surface of the RH domain in a late stages of autophagy, no structural information has been avail- mode that is distinct from those of other Rab–effector complexes. able for the RH domain of any of these proteins. Rubicon residues at the dimer interface are required for Rubicon We sought to determine the structural basis for Rab7-dependent and Rab7 to colocalize in living cells. Mutation of Rubicon RH res- recruitment of Rubicon with the long-term goal of blocking this step idues in the Rab7-binding site restores efficient autophagic flux in therapeutically. The structure revealed a four-zinc-cluster–based fold the presence of overexpressed Rubicon, validating the Rubicon RH for the Rubicon RH domain and revealed how Rubicon and Rab7 BIOPHYSICS AND COMPUTATIONAL BIOLOGY domain as a promising therapeutic target. interact. Rubicon mutants that disrupted the Rubicon:Rab7 in- teractionwereshowntomislocalizeandtobeincapableofsup- autophagy | Rab GTPase | crystal structure pressing autophagy, validating this interface as a potential drug target. acroautophagy (hereafter, “autophagy”) degrades bulky Results Mcytoplasmic materials by encapsulating them in a double Structure of the Rubicon RH Domain. We expressed a soluble 29.4- membrane structure, the autophagosome, and delivering them to kDa Rubicon RH domain construct comprising Rubicon residues the lysosome for degradation (1). Autophagy is critical for main- taining cellular homeostasis and functions as a response to events Significance such as starvation, pathogenic invasion, damage to organelles, and toxic protein aggregation (2). Failure of autophagy is associated Autophagy (cellular self-eating) is essential for the health and with aging, cancer, heart disease, neurodegeneration, and metabolic survival of eukaryotic cells. Therapeutic autophagy induction is disorders (3). Autophagy regulators are therefore key targets for a major goal in the field. Rubicon inhibits autophagy and is a the development of novel therapeutics (4, 5). Drugs that directly potential target for autophagy inducers. Rubicon is localized to up-regulate autophagy, whether by activation of a positive regulator its site of action in the cell by binding to the small GTPase Rab7. or inhibition of a negative regulator, are highly sought after. Here, we report a high-resolution structure of a large part of Rubicon (Run domain Beclin-1 interacting and cysteine-rich Rubicon, known as the Rubicon Homology (RH) domain. We containing) is a potent negative regulator of autophagy and show how the RH domain binds to Rab7 and show that the Rab7- – endolysosomal trafficking (6, 7). Rubicon disrupts autophagosome binding residues of Rubicon are essential for Rubicon localization lysosome fusion by inhibiting the class III phosphatidylinositol and autophagy inhibition. This provides a roadmap to block Ru- – 3-kinase complex 2 (PI3KC3-C2) (6 8). Rubicon is targeted to its bicon localization and activity in order to upregulate autophagy. sites of action by the late endosomal small GTPase Rab7 (9, 10). Autophagy suppression by Rubicon in aging has been linked to de- Author contributions: H.K.B., M.H., T.Y., and J.H.H. designed research; H.K.B., K.T., J.M.B., creased clearance of α-synuclein aggregates in neural tissues, im- D.P.F., I.A., F.D., and Y.J.I. performed research; D.P.F., I.A., and F.D. contributed new pairment of liver cell homeostasis, and interstitial fibrosis in the reagents/analytic tools; H.K.B., M.H., F.D., Y.J.I., and J.H.H. analyzed data; and H.K.B. kidney (11–13). Together with another target for autophagy-inducing and J.H.H. wrote the paper. Competing interest statement: The authors declare a competing interest. J.H.H. is a co- therapeutics, Bcl-2 (14, 15), Rubicon is one of very few known founder of Casma Therapeutics. broadly acting negative regulators of autophagy. Consistent with the This article is a PNAS Direct Submission. concept that decreased autophagic function is associated with aging, This open access article is distributed under Creative Commons Attribution-NonCommercial- Rubicon knockout increases lifespan in worms and flies (12). Most NoDerivatives License 4.0 (CC BY-NC-ND). encouragingly, Rubicon knockout mice show decreases in multiple α Data deposition: Structural coordinates generated in this work have been deposited in age-associated pathologies, including kidney fibrosis and -synuclein the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) accumulation (12). under accession code 6WCW. Rubicon consists of an N-terminal RUN domain with unknown 1Present Address: Biophysics Graduate Program, University of California, San Francisco, function, a middle region (MR) which contains its PI3K-binding CA 94115. domain (PIKBD) (8), and a C-terminal Rubicon homology (RH) 2To whom correspondence may be addressed. Email: [email protected]. domain which mediates interaction with Rab7 (9) (Fig. 1A). Rab7 is This article contains supporting information online at https://www.pnas.org/lookup/suppl/ the characteristic Rab marker of late endosomes (16). Rubicon is doi:10.1073/pnas.2008030117/-/DCSupplemental. the defining member of a class of metazoan RH domain-containing First published July 6, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2008030117 PNAS | July 21, 2020 | vol. 117 | no. 29 | 17003–17010 Downloaded by guest on September 23, 2021 699–949. It was essential to supplement the expression medium The overall structure of Rubicon RH has the shape of the with ZnCl2 to obtain measurable quantities of Rubicon. Rubicon letter “L,” where the C-terminal base of the “L” contacts Rab7, RH bound stably to Rab7–GTP in size exclusion chromatography and the stalk extends away toward its N terminus. The “L” is ∼80 Å (Fig. 1 B and C), where Rab7–GTP refers to a GTP-locked Q67L long by 50 Å wide. The main RH domain fold consists of four Zn mutant construct comprising residues 2–183. The C-terminal tail of clusters connected to each other by a helical stalk (Figs. 1D and Rab7, which ends in a dicysteine motif that is prenylated in vivo, 2A). Zn cluster 1 sits alone at the N-terminal end of the helical was truncated in this construct. We determined the structure of the stalk. Zn clusters 2–4 form a cluster of clusters at the C-terminal Rubicon RH–Rab7–GTP complex (Figs. 1D and 2A)byX-ray end of the stalk. Zn clusters 1 and 4 are Cys-4 (bound by four crystallography at a resolution of 2.8 Å. Phases were determined cysteines), and Zn 2 and 3 are Cys-2 His-2 (two Cys and two His) by molecular replacement with a search model based on contact clusters (Fig. 1 E and F). The latter three of the Zn clusters are prediction from coevolutionary data (18). The asymmetric unit of close to the Rab7-binding site (Fig. 1F), and the first is distal to thecrystalcontainsasingleRubiconRH–Rab7 complex. Statistics Rab7 (Fig. 1E). A pair of helices precedes the RH core, and a of the crystallographic data collection and structure refinement are single C-terminal helix follows (Fig. 2A). provided in SI Appendix,TableS1, and representative electron The overall Rubicon RH fold is unique, as determined by a density is displayed in Fig. 2B. structural query against the Protein Data Bank (PDB) using Dali ABC 1000 kDa 900 1 181 471 699 972 800 Rubicon:Rab7 Rubicon 50 700 Free Rab7 600 RUN PIKBD RH 37 500 400 Crystal Structure 1 207 25 Rubicon RH 300 (699-949) Void Rab7 20 200 Rab7 100 280nm Absorbance (mAU) 0 Crystal Structure 15 (2-183) 30 40 50 60 70 80 90 100 Retention Volume (mL) D 699 Rubicon Zn2+ 949 90 Mg2+ Rab7 GTP C881 H877 E F Zn 2 H907v C884 Zn 3 C909 C721 C739 H920 Zn 1 C826 C894 C923 C891 C724 H818 H751 C918 H848 C912 C750 C749 C929 C915 Zn 4 C932 Zn 2 G Rubicon RH Rab7-GTP Rab7-GPPNP L73 L73 Zn 3 S72 S72 Q71 Zn 4 R69 E68 Fig.
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