Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

Six novel inhibitors of Vibrio harveyi chitinase A (VhChiA), a family-18 chitinase homolog, were identified by in vitro screening of a library of pharmacologically active compounds. Unlike the previously identified inhibitors that mimicked the reaction intermediates, crystallographic evidence from 14 VhChiA-inhibitor complexes showed that all of the inhibitor molecules occupied the outer part of the substrate-binding cleft at two hydrophobic areas. The interactions at the aglycone location are well defined and tightly associated with Trp-397 and Trp-275, whereas the interactions at the glycone location are patchy, indicating lower affinity and a loose interaction with two consensus residues, Trp-168 and Val-205. When Trp-275 was substituted with glycine (W275G), the binding affinity toward all of the inhibitors dramatically decreased, and in most structures two inhibitor molecules were found to stack against Trp-397 at the aglycone site. Such results indicate that hydrophobic interactions are important for binding of the newly identified inhibitors by the chitinase. X-ray data and isothermal microcalorimetry showed that the inhibitors occupied the active site of VhChiA in three different binding modes, including single-site binding, independent two-site binding, and sequential two-site binding. The inhibitory effect of dequalinium in the low nanomolar range makes this compound an extremely attractive lead compound for plausible development of therapeutics against human diseases involving chitinase-mediated pathologies.

Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12–ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12–ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12–ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation. Abbreviations : ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle’s balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5ʹ-[β,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5ʹ-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; K_d: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type