Deciphering the catalytic domain of colicin M, a peptidoglycan lipid II-degrading enzyme.
Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.
Université Paris-Sud 11, UMR 8619, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, 91405 Orsay.
Hélène
H
Barreteau
Ahmed
A
Bouhss
Fabien
F
Gérard
Denis
D
Duché
Boubekeur
B
Boussaid
Didier
D
Blanot
Roland
R
Lloubès
Dominique
D
Mengin-Lecreulx
Thierry
T
Touzé
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010457:Peptidoglycan / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D003087:Colicins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli