Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Abstract

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 fulllength 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 surfaceexposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…However, on the basis of truncation experiments, Barreteau et al reported that the minimum catalytically active region of colicin M corresponded to amino acids 122-271 and that helix 1 (residues 37-46) is required for receptor binding. This delineates the receptor binding domain as a compact bundle of five helices spanning residues 37-122 (residues 40 -127 in syringacin M) (40). As expected from the extensive sequence similarity (Fig.…”

“…4 The mechanism of lipid II cleavage by colicin M-like bacteriocins is currently unknown, and although our data do not provide a clear picture of this mechanism, some speculation is warranted. From mutational and structural studies of colicin M, two conserved aspartic acid residues (Asp-226 (Asp-232) and Asp-229 (Asp-235)), a histidine residue (His-235 (His-241)), and an arginine residue (Arg-236 (Arg-242)) have been identified as key catalytic residues (40,41). In the structure of colicin M, the Asp-226 side chain lies some distance from the other catalytic residues, which led to the proposal that the active site of colicin M may be highly elongated as a consequence of the size of the large lipid II substrate.…”

Toxicity of the Colicin M Catalytic Domain Exported to the Periplasm Is FkpA Independent

“…1). The sp-C 119 domain has been reported to be active in vitro (4). As indicated in Table 3, sp-RC and sp-C 119 are toxic to the producing cells, in contrast to the toxicities of sp-TR and sp-C 141 .…”

“…First, ColM has an unusual mode of action through causing lysis by inhibiting peptidoglycan synthesis. Although its enzymatic activity and three-dimensional crystal structure have been reported (11,29), the amino acids that comprise its active site have not yet been clearly identified (4). Second, the toxic activity of the imported ColM depends upon the presence of a specific periplasmic protein (FkpA) (14), a chaperone, and a cis-and trans-peptidylprolyl isomerase (PPiase) (2).…”

CbrA Is a Flavin Adenine Dinucleotide Protein That Modifies the Escherichia coli Outer Membrane and Confers Specific Resistance to Colicin M

“…We previously proposed that unfolding of Cma during import involves a trans to cis isomerization of a proline bond, which is then cis to trans isomerized by FkpA during Cma refolding in the periplasm (20). This proposal is supported by the inactivity of Cma mutants in which other amino acids substitute for one specific proline residue (20) located outside the Cma active center (3,20,33) and by the high rate of cis-trans isomerization of this proline bond built into a synthetic peptide by FkpA (20).…”