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, 13 (1), 70-81

The 1:2 Complex Between RavZ and LC3 Reveals a Mechanism for Deconjugation of LC3 on the Phagophore Membrane

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The 1:2 Complex Between RavZ and LC3 Reveals a Mechanism for Deconjugation of LC3 on the Phagophore Membrane

Do Hoon Kwon et al. Autophagy.

Abstract

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.

Keywords: ATG4B; LC3; Legionella pneumophila; RavZ; SAXS; crystal structure; xenophagy.

Figures

Figure 1.
Figure 1.
Interaction between RavZ and LC3. (A) The RavZ-LC3 complex (red line) and RavZ alone (blue line) analyzed by SEC-MALS. The horizontal line represents the measured MM. Each species is indicated by an arrow with experimental (MALS) and theoretically calculated (Calc) molar mass values shown in parentheses (MALS/Calc). This shows the 1:2 binding stoichiometry between RavZ and LC3. (B) Domain architecture and RavZ constructs in this study. Three potential LIR motifs in the primary sequence are numbered sequentially: LIR1, LIR2 and LIR3. The abbreviations of each construct and the calculated molecular weights are shown. (C) The SEC-MALS results with deletion mutants of RavZ in the presence of LC3. ΔN (magenta line), N-Cat (green line) and Mt-C (blue line) in the presence of LC3 elute earlier and their MMs determined by SEC-MALS are 15-kDa greater than the calculated MMs of the free mutants, indicating 1:1 complex formation. The ΔNΔC mutant comprising deletion of the flexible N- and C-terminal regions (brown line) is unable to form a complex with LC3. Excess LC3 with MM of 15 kDa elutes later. (D) Summary of SPR data. The binding of RavZ and its deletion mutants to immobilized LC3 was measured, and the binding of ATG4B was also measured as a test case (See Fig. S1A and S2 for the SPR sensorgrams). The equilibrium dissociation constant (KD) was obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka).
Figure 2.
Figure 2.
Structure of RavZ. (A) Ribbon diagram of RavZ from L. pneumophila. The catalytic (residues 49–325) and membrane targeting (residues 326–423) domains are colored aquamarine and yellow, respectively. The catalytic site is indicated by an arrow. The N-terminal 48 and C-terminal 71 residues are missing and shown as red and green dots, respectively. The secondary structural elements are labeled sequentially. (B) Schematic representation of full-length RavZ. The invisible N-terminal and C-terminal regions in the crystal structure are represented by a red and green dotted line, respectively. The putative LC3-binding sites, LIR1, LIR2 and LIR3, are marked. (C) Superposition of 2 crystal structures (crystal form I and III) showing different loop conformations near the active site. This view is re-oriented from Fig. 2A to show the catalytic triad (approximately 110° and 30° along the horizontal and vertical axes, respectively). The overall structure is virtually identical except for the active site region. (D) A close-up view of the active site, which is highlighted as a transparent box in panel (C). The active site is covered by the flexible internal loop in the closed conformation of crystal form I (cyan), whereas it is widely accessible in the open conformation of crystal form III (blue). Catalytic triad residues (His176, Asp197 and Cys258) are presented as stick models. The interacting atoms in the catalytic triad are lined green and yellow for the closed and open conformations, respectively. The movement of the flexible loop is indicated by a double-headed red arrow.
Figure 3.
Figure 3.
Sequence alignment of LIR motifs of RavZ and solution behavior of the mutants. (A) Sequence alignment of 3 potential RavZ LIR motifs, LIR1, LIR2 and LIR3. The critical hydrophobic phenylalanine and leucine (or isoleucine) residues in the F-x-x-L(I) motif are colored red and the preceding 2 acidic residues are colored green. (B) SEC-MALS results confirming the importance of the LIR motifs. Green, red and blue profiles represent mutLIR1, mutLIR2 and mutLIR3, respectively, in the presence of LC3, and the corresponding horizontal lines represent the measured data obtained by MALS. Each species is indicated by an arrow with experimental (MALS) and theoretically calculated (Calc) molar mass values shown in parentheses (MALS/Calc). All LIR mutants form a 1:1 complex and, taken together with the MALS results using deletion constructs (See Fig. 1C), both LIR1 and LIR2 motifs participate in the binding of one LC3, while LIR3 binds to one LC3, independently.
Figure 4.
Figure 4.
Accumulation of autophagosomes in RavZ mutant cells. (A) HEK293 cells were transduced with GFP-LC3. After 24 h of infection, cells were transfected with expression vectors for MYC-tagged RavZ WT (FL) and C258S inactive mutant as a control. After 24 h of transfection, cells were left untreated (-Rapa) or treated (+Rapa) with 500 nM rapamycin for 12 h. Cells were then fixed, permeabilized, and stained with DAPI. Representative images of GFP-LC3 (green) and DAPI (blue) fluorescence are shown. Scale bar: 10 μm. (B) The same experiments with RavZ variants (N-Cat, Mt-C, Cat-Mt-C, mutLIR1, mutLIR1 and mutLIR1). (C) The fluorescence intensity of GFP-LC3 in the cytoplasm of > 50 cells for each experimental group was quantified and expressed as a percentage relative to the control group. Data represent the means ± SEM (standard error of mean) from 3 independent experiments.
Figure 5.
Figure 5.
Structure of the 1:2 complex between RavZ and LC3. (A) Scattering curve of RavZ alone, (B) Distance distribution function, P(r), of RavZ alone and (C) Molecular envelope of RavZ alone. The high-resolution crystal structure of RavZ was fitted into the low resolution molecular envelope generated by the SAXS data by rigid body docking. (D) Scattering curve of the RavZ-LC3 complex, (E) Distance distribution function, P(r), of the RavZ-LC3 complex and (F) Molecular envelope of the RavZ-LC3 complex. The 1:2 LC3-RavZ-LC3 model was generated using Chimera (UCSF). Two molecules of LC3 are located in the direction of the invisible N- and C-terminal regions of RavZ.
Figure 6.
Figure 6.
A plausible model for the mode of action of RavZ. The RavZ protein is secreted from L. pneumophila and then targeted to the phagophore or autophagosomal membrane. The catalytic domain of RavZ possessing cysteine protease activity is colored cyan and the membrane-targeting domain possessing binding affinity for the PtdIns3P-rich membrane is colored orange. The LIR motif containing the N- and C-terminal regions are indicated as red and green dots, and labeled as Nt and Ct, respectively. PE-conjugated and membrane-anchored LC3 is shown as a blue ribbon and PE is shown as a red ball with tails. Cleaved LC3 (by RavZ) is shown in yellow. RavZ possesses higher binding affinity for LC3 than ATG4B because it has 2 independent LIR motifs at the N- and C-terminal regions (Nt and Ct, respectively). We propose that RavZ achieves greater binding affinity for membrane-bound LC3 molecules (blue ribbon) by using the N- and C-terminal LIR motifs, and this event is critical for correct orientation to facilitate cleavage of the LC3–PE substrate generating a product that cannot be reconjugated to PE (yellow ribbon). Using this elegant and superior mechanism with RavZ as the key player, L. pneumophila has evolved an effective survival mechanism against host cell autophagy.

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