Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae
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Evaluation Summary:
Proteins that form pores in biological membranes are found in diverse contexts, including pathogenic toxins that help infect or lyse target cells and organelles. The study by Nadeem et al. reports on the properties of an α-pore-forming toxin, MakA, produced by the human pathogen, V. cholerae. The study is a remarkable example of a pH-induced structural mechanism of membrane remodeling. The insights reported here will be of interest to a wide range of scientists studying host-pathogen interactions, membrane remodeling, and macromolecular structure.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae . As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.
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Evaluation Summary:
Proteins that form pores in biological membranes are found in diverse contexts, including pathogenic toxins that help infect or lyse target cells and organelles. The study by Nadeem et al. reports on the properties of an α-pore-forming toxin, MakA, produced by the human pathogen, V. cholerae. The study is a remarkable example of a pH-induced structural mechanism of membrane remodeling. The insights reported here will be of interest to a wide range of scientists studying host-pathogen interactions, membrane remodeling, and macromolecular structure.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The authors provide strong evidence for how MakA interacts with membranes. The structural studies are, in general, sound, and provide a high resolution picture of how MakA undergoes a pH-dependent conformational change.
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Reviewer #2 (Public Review):
Prior work identified the V. cholera MakA/B/E tripartite toxin. The present study reports, however, that even in isolation the MakA subunit alone has pore-forming and cell-killing properties. This activity depended on acidic pH; above neutral pH MakA is soluble. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms high molecular-weight oligomers, binds and remodels membranes into high-curvature tubes, and eventually leads to loss of membrane integrity. Especially exciting, the authors report a helical reconstruction of MakA filaments with lipids that appears unprecedented. Nadeem and co-authors succeeded in showing us how low pH leads MakA to refold and embed a hairpin of transmembrane helices into a distorted, thinned, and discontinuous lipid bilayer. The conclusions …
Reviewer #2 (Public Review):
Prior work identified the V. cholera MakA/B/E tripartite toxin. The present study reports, however, that even in isolation the MakA subunit alone has pore-forming and cell-killing properties. This activity depended on acidic pH; above neutral pH MakA is soluble. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms high molecular-weight oligomers, binds and remodels membranes into high-curvature tubes, and eventually leads to loss of membrane integrity. Especially exciting, the authors report a helical reconstruction of MakA filaments with lipids that appears unprecedented. Nadeem and co-authors succeeded in showing us how low pH leads MakA to refold and embed a hairpin of transmembrane helices into a distorted, thinned, and discontinuous lipid bilayer. The conclusions are supported by the data and I have only minor comments for the authors to consider in preparing a final version for publication.
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Reviewer #3 (Public Review):
The main strength of the manuscript is the structural analysis of the MakA-membrane nanotubes, which corresponds to a new type of supramolecular assembly with a very unsual lipid and protein distribution. While the authors provide solid evidence about the concentration- and pH-depdendence of the induction of these protein/lipid nanotubes by the addition of MakA, the main weakness of the study lies in the fact that two main issues question the phyisiological relevance of these structures. First, the topology of the protein with respect to the lipid bilayer is opposite to the one that the protein would encounter upon endocytosis and acidification in the lysosomes. Meaning the protein in this case would be inside the lysosomes and not outside. Second, if the protein were to be secreted by the pathogen into the …
Reviewer #3 (Public Review):
The main strength of the manuscript is the structural analysis of the MakA-membrane nanotubes, which corresponds to a new type of supramolecular assembly with a very unsual lipid and protein distribution. While the authors provide solid evidence about the concentration- and pH-depdendence of the induction of these protein/lipid nanotubes by the addition of MakA, the main weakness of the study lies in the fact that two main issues question the phyisiological relevance of these structures. First, the topology of the protein with respect to the lipid bilayer is opposite to the one that the protein would encounter upon endocytosis and acidification in the lysosomes. Meaning the protein in this case would be inside the lysosomes and not outside. Second, if the protein were to be secreted by the pathogen into the extracellular medium of the host, it is unclear that the pH could be acidic and thereby enable the conditions used in this study.
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