Phosphoinositide-mediated oligomerization of a defensin induces cell lysis
- La Trobe University, Australia
Figures
Figure 1 with 1 supplement
Interaction of NaD1 with lipids.
(A) Detection of NaD1 binding to cellular lipids by protein-lipid overlay assay. Blots are representative of at least two independent experiments for each strip. (B) Binding of NaD1 to PIP2-containin…
Figure 1—figure supplement 1
Relative binding of NaD1 to lipids.
Quantitation by densitometry of the relative binding of NaD1 to lipids (normalized to PtdIns(4,5)P2 in A and B, sulfatide in C) on a (A) Membrane Lipid Strip, (B) PIP Strip, and (C) SphingoStrip. …
Figure 2
Crystal structure of the NaD1:PIP2 complex.
(A) Two orthogonal views of a cartoon representation of the NaD1:PIP2 oligomer comprising 14 NaD1 monomers (shown as ribbons) and 14 PIP2 molecules (shown as green sticks). The surface of the NaD1 …
Figure 3 with 2 supplements
Detailed view of the crystal structure of the NaD1:PIP2 complex.
In all panels, hydrogen bonds and salt bridges are shown as black dotted lines. (A) View of the interface of two NaD1 monomers revealing the hydrogen bonding pattern, with monomer I shown in cyan …
Figure 3—figure supplement 1
Cartoon of two NaD1 dimers with four bound PIP2 molecules.
https://doi.org/10.7554/eLife.01808.008
Figure 3—figure supplement 2
Simulated anneal omit map of a single PIP2 molecule bound to an NaD1 dimer, contoured at 1σ.
https://doi.org/10.7554/eLife.01808.009
Figure 4
The dimeric NaD1 ‘cationic grip‘ with two bound PIP2 molecules.
(A) Surface view in two orientations of a NaD1 dimer (monomer I in cyan and monomer II in magenta) with two bound PIP2 molecules (yellow and green). (B) The same as in A except that the surface …
Figure 5
NaD1 forms oligomers with PIP2.
(A) Ability of NaD1 to form multimers in the presence of PIP2 as determined by protein–protein cross-linking with BS3 followed by SDS-PAGE and Coomassie Brilliant Blue staining. (B) TEM of NaD1:PIP2 …
Figure 6 with 1 supplement
Multimerization of the NaD1:PIP2 complex.
(A) Schematic representation of residues from neighboring NaD1 monomers involved in binding two PIP2 molecules. Ability of rNaD1, rNaD1(R40E), and rNaD1(I37F) to (B) bind cellular lipids by …
Figure 6—figure supplement 1
Relative binding of rNaD1, rNaD1(I37F), and rNaD1(R40E) to lipids.
Quantitation by densitometry of the relative binding (normalized to PtdIns(4,5)P2) of (A) rNaD1, (B) rNaD1(I37F), and (C) rNaD1(R40E) to lipids on PIP Strips. Data shown as mean ± SD (n = 2) for A …
Figure 7 with 3 supplements
NaD1 kills mammalian tumor cells by membrane permeabilization.
(A) Forward scatter, side scatter, and PI uptake analysis of U937 cells treated with NaD1. (B) Binding of FITC-dextran and (C) LDH release by NaD1-treated U937 cells. Error bars in C indicate SEM (n …
Figure 7—figure supplement 1
NaD1 rapidly permeabilizes U937 cells.
Ability of NaD1 (0.5–20 μM) to mediate the release of ATP from U937 cells was examined using the ATP bioluminescence assay. The energy-dependency of the light-emitting luciferase-catalyzed oxidation …
Figure 7—figure supplement 2
Reduced and alkylated NaD1 (NaD1R&A) does not permeabilize U937 cells.
Ability of NaD1R&A to permeabilize U937 cells was investigated using the (A) PI uptake, (B) LDH release, and (C) ATP bioluminescence assays, with native NaD1 being included as a positive control. …
Figure 7—figure supplement 3
Tumor/transformed cells are more susceptible to killing by NaD1 than normal/primary cells.
MTT cell viability assays were performed on the indicated cell lines. The IC50 values are shown as mean ± SD (n≥ 2).
Figure 8 with 2 supplements
NaD1 induces membrane blebbing of tumor cells.
(A) CLSM of PKH67-stained NaD1 (10 μM) permeabilized HeLa and U937 cells. (B) CLSM of U937 cells treated with NaD1 (20 μM) in the presence of PI and 4 kDa FITC-dextran. Arrows indicate entry of PI. …
Figure 8—figure supplement 1
NaD1-induced membrane blebs do not retract once U937 cells are permeabilized.
Live confocal laser scanning microscopy (CLSM) of NaD1-treated U937 cells in the presence of PI. Cells were imaged over a period of 40 min, with NaD1 (10 μM) being added at 7 min.
Figure 8—figure supplement 2
NaD1-mediated membrane permeabilization occurs at the blebs of PC3 cells.
Live CLSM of PC3 cells treated with NaD1 in the presence of PI and 4 kDa FITC-dextran. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (20 μM), and 4 kDa FITC-dextran (100 μg/ml) …
Figure 9
Subcellular localisation of BODIPY-NaD1 in tumor cells.
(A) Detection of BODIPY-NaD1 binding to viable and permeabilized U937 cells by flow cytometry. (B) CLSM of subcellular localization of BODIPY-NaD1 (10 μM) on permeabilized U937, PC3, and HeLa cells. …
Figure 10
Expression of GFP-PH(PLCδ) in HeLa cells significantly delays NaD1-mediated cell permeabilization compared with cells expressing free GFP.
(A) CLSM of NaD1 (10 μM) treated HeLa cells expressing GFP-PH(PLCδ). Scale bars represent 10 μm. (B) The average length of time taken for NaD1 (10 μM) to permeabilize (PI-positive) …
Figure 11
Permeabilization of U937 cells is impaired in rNaD1(R40E).
Ability of rNaD1, rNaD1(R40E) and rNaD1(I37F) to permeabilize U937 cells as assessed by (A) PI uptake, (B) LDH release, and (C) FITC-dextran binding assays. Error bars in A and B indicate SEM (n = …
Figure 12
PIP2 fit into the NaD1 ‘cationic grip’.
Cut-away of PIP2 bound in the NaD1 cationic grip. NaD1 dimer surface is shown in gray, PIP2 in green and select NaD1 residues are shown as stick representation. NaD1 monomeric chains are colored in …
Figure 13
Proposed molecular mechanism of NaD1-mediated tumor cell lysis.
Schematic representation of NaD1-induced membrane blebbing and permeabilization. The assembly of NaD1:PIP2 oligomer can potentially be formed by (i) sequential recruitment of a NaD1 monomer followed …
Videos
Video 1
NaD1 rapidly induces membrane blebbing and permeabilization of HeLa cells.
Live CLSM of PKH67-stained HeLa cells in the presence of PI. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (10 μM) being added to cells at 1 min.
Video 2
Formation of a single large membrane bleb on a U937 cell following NaD1 treatment.
Three-dimensional reconstruction of CLSM images of a NaD1-treated (10 μM) PKH67-/PI-stained U937 cell.
Video 3
NaD1-mediated membrane permeabilization occurs at the blebs of U937 cells.
Live CLSM of U937 cells treated with NaD1 in the presence of PI and 4 kDa FITC-dextran. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (20 μM) and 4 kDa FITC-dextran (100 μg/ml) …
Video 4
BODIPY-NaD1 accumulates at the plasma membrane and certain intracellular organelles.
Three-dimensional reconstruction of CLSM images of BODIPY-NaD1-treated (10 μM) PI-stained PC3 cells.
Video 5
BODIPY-NaD1 accumulates at the plasma membrane and certain intracellular organelles.
Three-dimensional reconstruction of CLSM images of BODIPY-NaD1-treated (10 μM) PI-stained HeLa cells.
Video 6
Expression of GFP-PH(PLCδ) in HeLa cells delays NaD1-mediated cell permeabilization.
Live CLSM of GFP-PH(PLCδ) transfected HeLa cells treated with NaD1 in the presence of PI. Cells were imaged over a period of 15 min (5 s/frame), with NaD1 (10 μM) being added to cells at 30 s.
Video 7
Expression of free GFP in HeLa cells does not delay NaD1-mediated cell permeabilization.
Live CLSM of free GFP transfected HeLa cells treated with NaD1 in the presence of PI. Cells were imaged over a period of 20 min (5 s/frame), with NaD1 (10 μM) being added to cells at 30 s.
Tables
Table 1
Data collection and refinement statistics
| NaD1:PIP2 native | |
|---|---|
| Data collection | |
| Space group | C2221 |
| Cell dimensions | |
| a, b, c (Å) | 79.64, 132.04, 153.01 |
| α, β, γ (°) | 90.00, 90.00, 90.00 |
| Wavelength (Å) | 0.9537 |
| Resolution (Å)* | 40.84–1.6 (1.69–1.60) |
| Rsym or Rmerge* | 0.092 (0.617) |
| I/σI* | 11.6 (2.2) |
| Completeness (%)* | 99.7 (94.7) |
| Redundancy* | 6.7 (5.4) |
| Refinement | |
| Resolution (Å) | 40.37–1.6 |
| No. reflections | 105745 |
| Rwork/Rfree | 0.155/0.184 |
| No. atoms | |
| Protein | 10326 |
| Ligand/ion | 845 |
| Water | 816 |
| B-factors | |
| Protein | 21.5 |
| Ligand/ion | 28.9 |
| Water | 31.2 |
| R.m.s. deviations | |
| Bond lengths (Å) | 0.010 |
| Bond angles (°) | 1.651 |
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*
Values in parentheses are for highest resolution shell.
