The Legionella Anti-autophagy Effector RavZ Targets the Autophagosome via PI3P- and Curvature-Sensing Motifs

Abstract

Autophagy is a conserved membrane transport pathway used to destroy pathogenic microbes that access the cytosol of cells. The intracellular pathogen Legionella pneumophila interferes with autophagy by delivering an effector protein, RavZ, into the host cytosol. RavZ acts by cleaving membrane-conjugated Atg8/LC3 proteins from pre-autophagosomal structures. Its remarkable efficiency allows minute quantities of RavZ to block autophagy throughout the cell. To understand how RavZ targets pre-autophagosomes and specifically acts only on membrane-associated Atg8 proteins, we elucidated its structure. Revealed is a catalytic domain related in fold to Ulp family deubiquitinase-like enzymes and a C-terminal PI3P-binding module. RavZ targets the autophagosome via the PI3P-binding module and a catalytic domain helix, and it preferentially binds high-curvature membranes, intimating localization to highly curved domains in autophagosome intermediate membranes. RavZ-membrane interactions enhance substrate affinity, providing a mechanism for interfacial activation that also may be used by host autophagy proteins engaging only lipidated Atg8 proteins.

Figure 1. RavZ N-terminal domain is structurally homologous to the Ulp family of Ubl deconjugating enzymes

(A) RavZ domain organization and crystallization construct. (B) RavZ deconjugation activity assay. GL-1-PE (10 μM) on 25 nm liposomes (DOPE:POPC:blPI in a 55:35:10 molar ratio) was incubated for 1 h at room temperature with RavZ_WT or RavZ mutants (0.02 to 200 nM). The amount of deconjugated GL-1 relative to total GL-1 was quantified. Depicted are mean and standard deviation of three independent experiments. (C) Crystal structure of RavZ (residues 10-458, Δ23-43, Δ430-440) in ribbon representation colored blue to red from N- to C-terminus. The dotted line indicates boundary between N and C-terminal domains. (D) Secondary structure based superimposition (rmsd: 3.06 Å) of RavZ N-terminal domain (light blue) and protease domain of Ulp-family protein NEDP1 (green, PDB 2BKR). (E) RavZ (light blue) active site superposed with the NEDP1 (green, PDB 2BKR) active site. Residues numbered according to RavZ. (F) Model of the LC3 C-terminus (dark blue, ribbon representation, PDB 2Z0D) bound to the RavZ (light blue, surface representation) substrate-binding groove. RavZ/LC3 complex was generated by superimposing RavZ and LC3 onto the NEDP1/NEDD8 complex structure (PDB 2BKR). See also Figure S1.

Figure 2. The RavZ C-terminal domain includes a PI3P binding module responsible for RavZ autophagosomal localization

(A, C and D) Liposome flotation assays. The amount of liposome-bound RavZ relative to input RavZ was quantified based on SDS-PAGE analysis of fractions. Mean and standard deviation (SD) of three independent experiments are indicated. In representative SDS-PAGE gels the input lane contains 5% of total input protein and sample lanes contain 12.5% of total liposome bound protein. (A) RavZ_WT binding to liposomes (160 nm diameter) of indicated composition. Mean and SD are indicated; *P < 0.0001 compared to control (90% PC, 10% PE). For full statistical analysis see Table S2, tab 1. (B) Electrostatic surface potential of RavZ (± 5 kBT/e). Basic residues in putative PI3P binding site are indicated. (C) Binding of RavZ mutants to 160 nm diameter liposomes of indicated composition. *P < 0.001 as compared to RavZ_WT. For full statistical analysis see Table S2, tab 2. (D) Binding of RavZ mutants to liposomes of different diameters with 5% PI3P (DOPE:POPC:PI3P in a 30:65:5 molar ratio) and without (DOPE:POPC:blPI in a 30:60:10 molar ratio). Selected significant results are marked; *P < 0.01. For full statistical analysis see Table S2, tab 3. (E) RavZ deconjugation activity assay. Liposomes containing GL-1-PE (10 μM) were incubated for 1 h at room temperature with RavZ_WT or RavZ mutants (2 nM). Liposome composition was identical to (D) to ensure comparability. The amount of deconjugated GL-1 relative to total GL-1 was quantified. Mean and standard deviation (SD) of three independent experiments are indicated. *P < 0.01; compared to RavZ_WT on 100nm liposomes without PI3P. For full statistical analysis see Table S2, tab 4. (F) Delipidation assay as in (E), but with liposomes containing Ni-NTA lipid (65% POPC, 30%, DOPE, 5% DGS-NTA). Activity of C-terminally hexa-histidine tagged RavZ constructs tethered to the liposomes is compared to soluble, untagged versions of same constructs. The amount of deconjugated GL-1 relative to total GL-1 was quantified, and mean and SD of three independent experiments are shown. See also Figures S2 and S3.

Figure 3. RavZ requires both the PI3P binding domain and α3 for autophagosome localization and activity in vivo

(A) In vivo localization of RavZ_WT, RavZ_{R343A,K359A,K362A} and RavZ_Δ211-217 in HeLa cells, demonstrating that the residues in the putative PI3P-binding pocket and α3 are important for RavZ autophagosomal targeting. Localization of RavZ and the Atg16 autophagosome marker are shown in the first and second column, respectively. The third column shows the extent of their co-localization. (B) Images show LC3 (green) distribution in mouse bone marrow-derived macrophages infected with the indicated Legionella strains (red). (C) Percentage of LC3-puncta positive cells calculated from three independent assays, where a total of 100 cells were scored in each assay. Presented are the mean and SD; *P < 0.0001 compared to RavZ_WT. For full statistical analysis see Table S3.

Figure 4. Model of RavZ dependent Atg8/LC3 delipidation at the autophagosomal membrane

RavZ (dark blue) is recruited to the autophagosomal membrane (grey) by interaction of its C-terminal domain with PI3P (yellow). Helix 3 in the N-terminal protease domain likely interacts with the membrane by insertion of aromatic side chains between the lipid acyl-chains and by electrostatic interactions between its basic side chains and the negatively charged lipid headgroups. The RavZ N-terminal domain recognizes Atg8-PE/LC3-PE (Atg8/LC3: light blue, PE: green) at the membrane and catalyzes its deconjugation. The interaction of Helix 3 of the RavZ N-terminal domain with membrane is critical for efficient catalysis, possibly as a “lid” clamping the C-terminus of Atg/LC3 between RavZ and the membrane to restrict LC3 mobility. RavZ/LC3 complex was generated by superimposing RavZ and LC3 (PDB 2Z0D) onto the NEDP1/NEDD8 complex structure (PDB 2BKR).