Cryo-EM structure revealed a novel F-actin binding motif in a Legionella pneumophila lysine fatty acyltransferase
- Weill Institute for Cell and Molecular Biology, Cornell University, United States
- Department of Molecular Biology and Genetics, Cornell University, United States
- Department of Chemistry, Department of Molecular Biology and Genetics, Cornell University, United States
- Howard Hughes Medical Institute; Department of Medicine and Department of Chemistry, The University of Chicago, United States
Figures
Figure 1 with 1 supplement
Identification of LFat1 (lpg1387) as an F-actin binding effector.
(A) AlphaFold-predicted structure of Lfat1 (left) and domain architecture of Lfat1 (right) with the N-terminal domain shown in red, the C-terminal domain in pink, and the central coiled-coil domain in cyan. FL: full-length, NC: N and C globular domain, CC: coiled-coil domain, respectively. (B) Cellular localization of Lfat1-FL, -NC, or -CC as determined by fluorescence microscopy. HeLa cells transiently expressing GFP-fused Lfat1-FL, -NC, or -CC were fixed and stained with phalloidin. Scale bar = 10 µm. (C) Colocalization was determined by fluorescence intensity line scan along the yellow line shown in (B). Red = F-actin, green = GFP. (D) Co-immunoprecipitation to determine interaction of Lfat1 with actin. HEK293T cells transiently expressing either GFP-empty vector, -Lfat1 FL, -Lfat1 NC, or -Lfat1 CC were lysed, and cell lysates were immunoprecipitated using anti-GFP nanobeads. The IP samples were analyzed with SDS-PAGE followed by immunoblot against GFP and actin. (E) Co-sedimentation assay to determine direct interaction between Lfat1 CC and F-actin. Purified G-actin, CC, or G-actin plus CC was incubated either in G-actin buffer or F-actin polymerization buffer, then ultracentrifuged to separate supernatant from pellet, followed by analysis via SDS-PAGE. S: supernatant, P: pellet. (F) Binding stoichiometry between Lfat1 CC and actin as determined by co-sedimentation assay.
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Figure 1—source data 1
Original western blots and SDS-PAGE Coomassie staining gels displayed in Figure 1D, E, and F.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig1-data1-v1.zip
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Figure 1—source data 2
PDF files of original western blots and SDS-PAGE Coomassie staining gel displayed in Figure 1D, E, and F with labels.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig1-data2-v1.zip
Figure 1—figure supplement 1
Intracellular localization of representative Legionella effectors.
Individual plasmid from a library consisting of 315 GFP-tagged L. pneumophila effectors was transiently expressed in HeLa cells followed by PFA fixation, stained with rhodamine-phalloidin, and visualized by fluorescence microscopy. Images showing intracellular localization of several representative effectors: lpg0284 (nuclear); lpg1387 and MavH (F-actin); lpg1578 (ER); and lpg1803 (mitochondrial). Scale bar = 10 µm.
Figure 2 with 3 supplements
Cryo-electron microscopy (Cryo-EM) structure of the Lfat1 ABD in complex with F-actin.
(A) Cryo-EM structure of the F-actin-Lfat1 ABD complex. Left: Side view of the structure positioned with the pointed end (-) up and barbed end (+) down. The visible part of ABD is colored in cyan. Right: Top view of the complex. (B) The D-loop conformation and its interactions in the hydrophobic cleft of the n+2nd actin monomer. (C) Ribbon diagram of the distal portion of the Lfat1 coiled-coil domain. The two α-helices are zipped together through extensive hydrophobic interactions contributed mainly by leucine and isoleucine residues (shown in sticks). (D) Structural representation of the interaction between the ABD domain of Lfat1 and F-actin. Inset: Extensive hydrophobic, hydrogen bonding, and electrostatic interactions were observed between the Lfat1 ABD domain and the two adjacent actin monomers (for details, see in the text).
Figure 2—figure supplement 1
Cryo-electron microscopy (Cryo-EM) data processing details of F-actin-Lfat1ABD complex.
(A) Representative Cryo-EM micrograph of Lfat1ABD-F-actin complex. (B) Data-processing and structure determination workflow. Movies were collected at the Talos Arctica electron microscope. Motion correction and patch CTF estimation were performed using CryoSPARC. Following manual curation, automatic filament tracer-based particle picking, and extraction, 1.2 million particles were 2D classified and an ab initio model was built and refined using helical refinement until convergence. (C) Angular distribution of particles. (D) Resolution estimation by GSFSC. (E) Local resolution map of the final Cryo-EM map.
Figure 2—figure supplement 2
Cryo-electron microscopy (Cryo-EM) maps of the F-actin-Lfat1ABD complex.
(A) Final Cryo-EM density map of the complex. Top: Side view of the complex density map with the pointed end (-) up and barbed end (+) down. Lfat1 ABD is shown in cyan. Bottom: Top view of the complex density map. (B) The EM density map (gray) of ADP-Mg2+ at the ATP binding cleft of actin subunits. (C) The EM map of actin D-loop (yellow). (D) The EM density map (cyan) of the distal region of the Lfat1 hairpin.
Figure 2—figure supplement 3
The interface between Lfat1 ABD and F-actin.
(A) Surface representative of two adjacent actin molecules (purple and pink), and the D-loop region of the nth actin molecule is colored yellow. Lfat1 ABD is represented as a ribbon in cyan. Key residues involved in F-actin interaction are shown in sticks. (B) The interface between Lfat1 ABD and F-actin at the same orientation as in (A). The surface of the two actin molecules is colored based on hydrophobicity, with hydrophobic areas in orange and hydrophobic regions in pink.
Figure 3
Validation of key Lfat1 ABD residues in their contributions to F-actin interactions.
(A) Schematic diagram of the Lfat1 ABD domain. Three key residues (R236, Y240, and Q254) involved in actin binding are labeled. (B and C) F-actin localization analysis of indicated ABD mutants. GFP-Lfat1 WT, R236A, Y240A, or Q254A mutant was transiently expressed in HeLa cells followed by fixation and staining with phalloidin. Fluorescence images were taken by a confocal microscope and analyzed using line scan along the indicated yellow lines. Scale bar = 10 µm. (D) Co-sedimentation assay of F-actin with wild-type (WT) and mutant Lfat1 ABD. Increasing amounts (0–60 µM) of recombinant WT or mutant ABD proteins were incubated with a fixed amount of actin. The samples were ultracentrifuged after 30 min of room temperature incubation in 1× actin polymerization buffer. The supernatant and pellet fractions were analyzed by SDS-PAGE. (E) Quantitative analysis of the co-sedimentation titration data. The data point for each concentration was averaged from three technical replicates. The error bar represents the standard deviation.
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Figure 3—source data 1
Original SDS-PAGE Coomassie staining gel displayed in Figure 3D.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig3-data1-v1.zip
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Figure 3—source data 2
PDF files of original SDS-PAGE Coomassie staining gels displayed in Figure 3D with labels.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig3-data2-v1.zip
Figure 4
Structural comparison between Lfat1 and other ABD-F-actin complexes.
(A) Ribbon diagram (upper row) and surface representation (bottom row) of two adjacent actin monomers (purple and pink) bound with Lfat1 ABD, LifeAct (PDB ID: 7BTE), Utrophin (6M5G), and ExoY (7P1G). The region interfacing with each ABD is colored in cyan. (B) Structural comparison of the D-loop conformation in the Lfat1 ABD-F-actin complex (purple) with the D-loop in other F-actin-ABD complexes: F-actin alone (yellow), LifeAct (cyan), Utrophin (green), and ExoY (navy blue). Two D-loop residues with the largest deviation of backbone dihedral angles (G50 and Q51) are shown in sticks.
Figure 5 with 1 supplement
Engineering Lfat1 actin-binding domain (ABD) as an in vivo F-actin probe.
(A) Mapping the minimal ABD of Lfat1. Schematic of shortened Lfat1ABD fragments used for F-actin binding (Left). Representative fluorescence images of cells expressing indicated ABD fragments and stained with rhodamine-phalloidin. (B) Line-scan analysis for the indicated ABD probes along the yellow lines. (C) Representative fluorescence images of cells transiently transfected with plasmids expressing separated α-helices (mCherry-α1 and α2-GFP) of ABD, ABD-S1, and ABD-S2. The cells were fixed and stained with CF647-phalloidin. (D) Line-scan analysis of the images shown in (C). Scale bar = 10 µm.
Figure 5—figure supplement 1
Intracellular localization of separated individual α-helices of the Lfat1 actin-binding domain (ABD).
(A) Representative images of HeLa cells transiently expressing individual α-helices from full-length and truncated Lfat1 ABD. Cells were fixed and stained with CF647-phalloidin. (B) Line-scan analysis of constructs tested in (A). Scale bar = 10 µm.
Figure 6 with 2 supplements
Lfat1 is a lysine fatty acyltransferase that modifies eukaryotic small GTPases.
(A) Ribbon representation of the V. vulnificus Rho Inactivation Domain (RID) (PDB ID: 5XN7) catalytic domain (purple) superimposed with the AlphaFold-predicted NC-domain of Lfat1 (N-terminal domain in red, C-terminal domain in pink). Inset: the catalytic pockets of the two proteins with conservation of the catalytic histidine and cysteine between RID and Lfat1 shown in sticks. (B) Lfat1 catalyzes lysine fatty acylation of Rac3. N-terminal Flag-tagged Rac3 was co-expressed with either GFP empty vector (EV), GFP-Lfat1 WT, H38A, or C403A mutant in HEK293T cells for 24 hr. Flag-Rac3 was enriched using immunoprecipitation and subjected to click chemistry. The samples were then separated on SDS-PAGE and scanned for fluorescence signals.
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Figure 6—source data 1
Original western blots/gels corresponding to Figure 6B.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig6-data1-v1.zip
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Figure 6—source data 2
PDF files of original western blots/gels corresponding to Figure 6B with labels.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig6-data2-v1.zip
Figure 6—figure supplement 1
Identification of potential Lfat1 substrates by click chemistry-coupled Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) mass spectrometry.
(A) Flowchart for selecting high-confidence substrate hits from the SILAC screen. (B) Top hits of potential Lfat1 substrates identified in this SILAC-MS experiment. (C–F) Click chemistry verification of some small GTPases selected from the top hit list. N-terminal Flag-tagged RheB, RalA, RalB, or Rap1B was co-expressed along with GFP-Lfat1 or GFP control in HEK293T cells followed by anti-Flag immunoprecipitation and click chemistry conjugation reaction with an azide-containing fluorophore. The samples were separated on SDS-PAGE and scanned for fluorescence signals.
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Figure 6—figure supplement 1—source data 1
Original western blots/gels corresponding to Figure 6—figure supplement 1C, D, E, and F.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
PDF files of original western blots/gels corresponding to Figure 6—figure supplement 1C, D, E, and F with labels.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
The lysine fatty acyltransferase activity of Lfat1 does not depend on actin binding.
Lfat1 catalyzes lysine fatty acylation of RheB (A) and Rac3 (B). N-terminal Flag-tagged RheB and Rac3 were co-expressed with either GFP-Lfat1 WT, Y240A, or Q254A mutant in HEK293T cells for 24 hr. Flag-RheB and Flag-Rac3 were enriched using immunoprecipitation and subjected to click chemistry. The samples were then separated on SDS-PAGE and scanned for fluorescence signals or analyzed by western blot.
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Figure 6—figure supplement 2—source data 1
Original western blots/gels corresponding to Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig6-figsupp2-data1-v1.zip
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Figure 6—figure supplement 2—source data 2
PDF files of original western blots/gels corresponding to Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/106975/elife-106975-fig6-figsupp2-data2-v1.zip
Author response image 1
Tables
Table 1
CryoEM Data collection, refinement and validation statistics.
| Microscope | FEI Talos Artica |
| Voltage (keV) | 200 |
| Defocus range (um) | –0.4 to –3.0 |
| Camera | K3 direct electron detector |
| Pixel size (Å) | 0.833 (super-resolution) |
| Total electron dose (e/Å2) | 40.68 |
| Exposure time (seconds) | 1.23 |
| Frames per movie | 50 |
| Number of images | 4548 |
| 3-D refinement statistics and helical symmetry | |
| Total number of particles | 1,220,462 |
| Resolution (Å) | 3.58 |
| Helical twist | –167 |
| Rise | 28 |
| Model composition and validation | |
| Non-hydrogen atoms | 34,740 |
| Protein residues | 4390 |
| Ligands | 10 Mg, 10ADP |
| RMSD | |
| Bond lengths(Å) | 0.25 |
| Bond angles (°) | 0.5 |
| B-factor (Å2) Protein | 58.87 |
| B-factor (Å2) Ligand (ADP) | 56.6 |
| MolProbity Score | 1.9 |
| Clashscore | 5 |
| Ramachandran plot:-Favored | 95 |
| Allowed | 5 |
| Outlier | 0 |
| PDB ID | 8VAA |
| EMDB Code | 43087 |
Additional files
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Supplementary file 1
List of constructs used in this study.
- https://cdn.elifesciences.org/articles/106975/elife-106975-supp1-v1.xlsx
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Supplementary file 2
List of primers used in this study.
- https://cdn.elifesciences.org/articles/106975/elife-106975-supp2-v1.xlsx
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Supplementary file 3
Forward Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) result.
- https://cdn.elifesciences.org/articles/106975/elife-106975-supp3-v1.xlsx
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Supplementary file 4
Reverse Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) result.
- https://cdn.elifesciences.org/articles/106975/elife-106975-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/106975/elife-106975-mdarchecklist1-v1.docx
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Cryo-EM structure revealed a novel F-actin binding motif in a Legionella pneumophila lysine fatty acyltransferase
eLife 14:RP106975.
https://doi.org/10.7554/eLife.106975.3
