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Published on: February 5, 2019
X-ray structure and site-directed mutagenesis analysis of the Escherichia coli colicin M immunity protein
Fabien Gérard1, Mark A Brooks, Hélène Barreteau
1Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, Université Paris-Sud, Orsay F-91405, France.
Abstract:
Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four β strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.
Insights
Colicin M immunity protein (Cmi) structure reveals its protective mechanism against the Colicin M toxin. Key residues and a disulfide bond are essential for Cmi
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Colicin M (ColM) is a toxin produced by Escherichia coli that inhibits bacterial cell wall synthesis.
- ColM-producing bacteria are protected by the Cmi immunity protein, but its mechanism remains unknown.
Purpose of the Study:
- To elucidate the structure and function of the Colicin M immunity protein (Cmi).
- To understand the molecular basis of ColM immunity.
Main Methods:
- X-ray crystallography was used to determine the Cmi protein structure.
- Site-directed mutagenesis and functional assays were employed to identify key residues.
- DsbA oxidase involvement in disulfide bond formation was investigated.
Main Results:
- The crystal structure of Cmi revealed a compact fold with four beta strands and four alpha helices.
- A disulfide bond between Cys31 and Cys107, along with C-terminal residues, was found to be crucial for Cmi's immunity function.
- DsbA oxidase was confirmed to be involved in forming the Cmi disulfide bond.
Conclusions:
- The Cmi structure provides insights into its protective role against ColM.
- Specific structural features, including the disulfide bond and C-terminal residues, are essential for Cmi activity.
- This study identifies conserved residues within the YebF family, suggesting broader functional implications.
