NRBF2 homodimerization by its coiled-coil domain strengthens association with the PtdIns3K complex mediated by the MIT domain to promote autophagy

The mammalian class III phosphatidylinositol-3-kinase complex (PtdIns3K) forms two biochemically and functionally distinct subcomplexes including the ATG14-containing complex I (PtdIns3K-C1) and the UVRAG-containing complex II (PtdIns3K-C2). Both subcomplexes adopt a V-shaped architecture with a BECN1-ATG14 or UVRAG adaptor arm and a PIK3R4/VPS15-PIK3C3/VPS34 catalytic arm. NRBF2 is a pro-autophagic modulator that specifically associates with PtdIns3K-C1 to enhance its kinase activity and promotes macroautophagy/autophagy. How NRBF2 exerts such a positive effect is not fully understood. Here we report that NRBF2 binds to PIK3R4/VPS15 with moderate affinity through a conserved site on its N-terminal MIT domain. The NRBF2-PIK3R4/VPS15 interaction is incompatible with the UVRAG-containing PtdIns3K-C2 because the C2 domain of UVRAG outcompetes NRBF2 for PIK3R4/VPS15 binding. Our crystal structure of the NRBF2 coiled-coil (CC) domain reveals a symmetric homodimer with multiple hydrophobic pairings at the CC interface, which is in distinct contrast to the asymmetric dimer observed in the yeast ortholog Atg38. Mutations in the CC domain that rendered NRBF2 monomeric led to weakened binding to PIK3R4/VPS15 and only partial rescue of autophagy deficiency in <i>nrbf2</i> knockout cells. In comparison, NRBF2 with its CC domain replaced by a dimeric Gcn4 module showed proautophagic activity comparable to wild type while NRBF2 carrying a tetrameric Gcn4 module showed further enhanced activity. We propose that the oligomeric state of NRBF2 mediated by its CC domain is critical for strengthening the moderate NRBF2-PIK3R4/VPS15 interaction mediated by its MIT domain to fully activate PtdIns3K-C1 and promote autophagy.<b>Abbreviations:</b> ATG: autophagy related; ATG14: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; dCCD: delete CCD; dMIT: delete MIT; Gcn4: general control nonderepressible 4; ITC: isothermal titration calorimetry; IP: immunoprecipitation; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MIM: MIT-interacting motif; MIT: microtubule interacting and trafficking; NMR: nuclear magnetic resonance; NRBF2: nuclear receptor binding factor 2; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WT: wild type.