On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium
- Micalis Institute, France
- Leibniz Institute of Polymer Research, Germany
- CNRS Université Paris-Saclay, France
- Institut Curie, France
Abstract
In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of MreB biochemical and polymerization properties in vitro remains limited and is mostly based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized protofilaments by electron microscopy and the first 3D-structure of MreB from a Gram-positive bacterium. We show that Geobacillus stearothermophilus MreB forms straight pairs of protofilaments on lipid surfaces in the presence of ATP or GTP, but not in the presence of ADP, GDP or non-hydrolysable ATP analogs. We demonstrate that membrane anchoring is mediated by two spatially close short hydrophobic sequences while electrostatic interactions also contribute to lipid binding, and show that the population of membrane-bound protofilament doublets is in steady-state. In solution, protofilament doublets were not detected in any condition tested. Instead, MreB formed large sheets regardless of the bound nucleotide, albeit at a higher critical concentration. Altogether, our results indicate that both lipids and ATP are facilitators of MreB polymerization, and are consistent with a dual effect of ATP hydrolysis, in promoting both membrane binding and filaments assembly/disassembly.
Data availability
Protein structures data have been deposited in PDB under the accession codes 7ZPT and 8AZG.
Article and author information
Author details
Ines Li de la Sierra-Gallay
Rut Carballido-Lopez
Funding
European Research Council (ERC-SG,311231)
- Rut Carballido-Lopez
European Research Council (ERC-CG,772178)
- Rut Carballido-Lopez
Agence Nationale de la Recherche (ANR-11-LABX0038)
- Aurélie Bertin
Agence Nationale de la Recherche (ANR-10-IDEX-0001-02)
- Aurélie Bertin
VolkswagenStiftung
- Lars D Renner
Agence Nationale de la Recherche (ANR-10-INSB-05-05)
- Sylvie Nessler
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Petra Anne Levin, Washington University in St. Louis, United States
Version history
- Preprint posted: October 20, 2022 (view preprint)
- Received: October 26, 2022
- Accepted: October 8, 2023
- Accepted Manuscript published: October 11, 2023 (version 1)
Copyright
© 2023, Mao et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 328
- Page views
-
- 117
- Downloads
-
- 0
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium
eLife 12:e84505.
https://doi.org/10.7554/eLife.84505
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.
