On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium
- Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, France
- Leibniz Institute of Polymer Research, and the Max-Bergmann-Center of Biomaterials, Germany
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, France
- Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, Sorbonne Université, 75005, France
Figures
Figure 1 with 2 supplements
Crystal structure of the apo protofilament of MreB from G. stearothermophilus.
(A) Crystal structure of apo MreBGs (PDB ID 7ZPT), colored by subdomains, superimposed on the crystal structure of apo MreBTm (PDB ID 1JCF), in beige. The sequence similarity between the two …
Figure 1—figure supplement 1
Multiple sequence alignment of MreB proteins from several bacteria.
The sequence of G. stearothermophilus (MreBGs) was aligned using Clustal-Ω at PRABI against the homologous MreB sequences of the Gram-positive bacterium B. subtilis (MreBBs, GenBank ID ATA60829.1) …
Figure 1—figure supplement 2
Calibration curve and typical size exclusion chromatography profiles of MreB.
(A) Typical size exclusion chromatography elution profiles of MreBGs. MreBGs (wild-type) was loaded on a HiLoad 16/600 Superdex 200 pg (GE healthcare) size exclusion column immediately after elution …
Figure 2 with 5 supplements
MreBGs forms double protofilaments in the presence of ATP and lipids.
(A) Polymerization of MreBGs into pairs of protofilaments depends on the presence of lipids and ATP. MreBGs was set to polymerize in standard conditions in the presence or absence of ATP and lipid …
Figure 2—figure supplement 1
Workflow for quantification of MreB polymers on TEM grids.
(A) Schematic drawing of a 300 mesh EM grid displaying 12 imaging localization widespread on the observation field. (B) EM images of typical fields of view presenting no polymers (Left, ‘-‘), low …
Figure 2—figure supplement 2
MreB presents limited capacity to form polymers in solution.
(A) Quantification of pairs of protofilaments of MreBGs shows limited polymerization in solution and in the absence of lipids. MreBGs was set to polymerize in standard conditions (ATP and 100 mM …
Figure 2—figure supplement 3
MreBGs polymers display a broad range of lengths and widths.
(A–D) Dual protofilaments of MreBGs observed on various fields of a single EM grid. Example of fields containing exclusively medium size polymers (>100 nm) (A); exclusively short polymers (<50 nm) (B…
Figure 2—figure supplement 4
2D averaging of negatively stained images of MreBGs dual protofilaments showing the symmetrical arrangement of monomers.
Displayed are the 21 classes of images generated by 2D image processing (alignment and classification from 1 554 individual raw images). Scale bar, 20 nm.
Figure 2—figure supplement 5
MreBGs polymers coat and distort liposomes.
Cryo-EM micrographs of 0.37 mg/mL liposomes made from E. coli lipid total extract, alone (A) or mixed with 1.34 µM (0,05 mg/mL) purified MreBGs in the presence of 2 mM ATP and 100 mM (B) or 500 mM (C…
Figure 3 with 3 supplements
Double protofilaments of Geobacillus MreB efficiently form in the presence of hydrolysable nucleotides.
(A) ATP and GTP promote efficient assembly of MreBGs polymers on a lipid surface. MreB (1.34 µM; 0,05 mg/mL) was incubated in the presence of either ATP, ADP, GTP, GDP, or the non-hydrolysable …
Figure 3—figure supplement 1
Effect of nucleotides, lipids and protein concentration on the polymerization of MreB.
(A) Size distribution of MreBGs double filaments set to polymerize in the presence of ATP or GTP (2 mM) and 500 mM KCl. Negative stained EM micrographs were analyzed using FIJI and the length of …
Figure 3—figure supplement 2
Sheet-like structures formed by MreB in solution.
MreB at high concentration (6.7 µm; 0.25 mg/ml) was set to polymerize in solution in the presence of 2 mM of various nucleotides (ATP, ADP, or AMP-PNP). (A). Negatively stained EM images of typical …
Figure 3—figure supplement 3
QCM-D experiments of MreBGs adsorption on supported lipid bilayers.
(A) Lipid bilayer formation on crystal with SiO2 layers. Supported lipid bilayers (SLBs) are formed by spontaneous rupture of adsorbed liposomes as indicated by frequency shifts (Δf, black solid …
Figure 4 with 5 supplements
in theThe N-terminus and the α2β7 hydrophobic loop of MreBGs promote membrane binding and polymerization on a lipid surface.
(A) Both the hydrophobic α2-β7 loop and the N-terminus sequence of MreBGs are required for efficient polymerization on a lipid monolayer. Frequency and density of polymer formation in high salt (500 …
Figure 4—figure supplement 1
Distribution of N-terminal amphipathic helices and hydrophobic sequences on MreBs proteins in the bacterial kingdom.
N-terminal sequences of MreB proteins from selected species across the bacterial kingdom were aligned using Clustal-Ω. The N-terminal sequences were analyzed for the presence of a putative α-helix …
Figure 4—figure supplement 2
The protruding hydrophobic subdomain IA of MreBGs is surrounded by a positively charged cluster.
(A). Electrostatic surface potential of an MreBGS monomer (PDB ID 7ZPT), facing the domains I (IA, down and IB, up) side. The view corresponds to a 90° rotation along a vertical axis compared to the …
Figure 4—figure supplement 3
Circular dichroism (CD) spectra showing similar folding of the wild-type and the deletion mutants of the α2-β7 loop (ΔGLFA), the N-terminus (ΔNter) or both domains (ΔNter + ΔGLFA) of recombinant MreBGs.
Figure 4—figure supplement 4
The amino-terminal sequence, the GLFA residues of the α2-β7 hydrophobic loop and electrostatic interactions mediate binding of MreB to a lipid surface.
(A) Quantification of dual protofilament formation by MreBGs wild type (WT) and the mutants of the N-terminus (ΔNter), the α2-β7 loop (ΔGLFA), or both domains (ΔNter+ ΔGLFA), in the presence of …
Figure 4—figure supplement 5
Crystal structure of MreBGs bound to ATP.
(A) Electron density of the ATP molecule bound to MreBGs. The Fo-Fc omit map calculated by omitting the nucleotide from the model is shown as a grey mesh contoured at 4σ. The nucleotide and the …
Figure 5 with 1 supplement
ATPase activity of MreBGs.
(A) The ATPase activity of MreBGs is stimulated in the presence of lipids. ATPase activity, measured by monitoring inorganic phosphate (Pi) release, of MreBGs at different concentrations (0.26–1.34 …
Figure 5—figure supplement 1
Effects of lipids, temperature and deletions on the hydrolytic activity of MreB.
(A) The ATPase activity of MreBGs is stimulated at high temperature. Release of Pi detected by malachite green assay for a range of MreBGs concentrations (0.26–1.34 µM) in the presence or absence of …
Figure 6
Model for ATP-driven MreBGs membrane binding and polymerization into pairs of filaments.
ATP hydrolysis stimulates MreBGs adsorption to lipids, possibly by promoting a conformational change that renders the hydrophobic α2-β7 loop and N-terminal protruding region prone for insertion into …
Author response image 1
Electron density map after refinement with the classical conformation of ATP-Mg.
The phosphate and the Mg ion are clearly in the negative Fo-Fc (in red) while the positive Fo-Fc map (in green) clearly coincide with our superimposed atypical conformation (in cyan).
Additional files
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Supplementary file 1
Data-collection and refinement statistics.
- https://cdn.elifesciences.org/articles/84505/elife-84505-supp1-v2.xlsx
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Supplementary file 2
List of polymerization conditions assayed.
- https://cdn.elifesciences.org/articles/84505/elife-84505-supp2-v2.xlsx
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Supplementary file 3
List of proteins used in this study.
- https://cdn.elifesciences.org/articles/84505/elife-84505-supp3-v2.xlsx
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Supplementary file 4
List of strains used in this study.
- https://cdn.elifesciences.org/articles/84505/elife-84505-supp4-v2.xlsx
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Supplementary file 5
List of oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/84505/elife-84505-supp5-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84505/elife-84505-mdarchecklist1-v2.docx
