Limits...
Crystal structure of colicin M, a novel phosphatase specifically imported by Escherichia coli.

Zeth K, Römer C, Patzer SI, Braun V - J. Biol. Chem. (2008)

Bottom Line: The novel phosphatase domain displays no sequence similarity to known phosphatases.The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins.The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, Tübingen, Germany.

ABSTRACT
Colicins are cytotoxic proteins secreted by certain strains of Escherichia coli. Colicin M is unique among these toxins in that it acts in the periplasm and specifically inhibits murein biosynthesis by hydrolyzing the pyrophosphate linkage between bactoprenol and the murein precursor. We crystallized colicin M and determined the structure at 1.7A resolution using x-ray crystallography. The protein has a novel structure composed of three domains with distinct functions. The N-domain is a short random coil and contains the exposed TonB box. The central domain includes a hydrophobic alpha-helix and binds presumably to the FhuA receptor. The C-domain is composed of a mixed alpha/beta-fold and forms the phosphatase. The architectures of the individual modules show no similarity to known structures. Amino acid replacements in previously isolated inactive colicin M mutants are located in the phosphatase domain, which contains a number of surface-exposed residues conserved in predicted bacteriocins of other bacteria. The novel phosphatase domain displays no sequence similarity to known phosphatases. The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins. The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains.

Show MeSH

Related in: MedlinePlus

A, comparison of the colicin M amino acid sequence with the sequences of predicted bacteriocins of Burkholderia amifaria MC40-6 (accession number AOTM72), Burkholderia cepacia (QOBIY7), Pseudomonas syringae pv. tomato (Q88A25), and Pseudomonas aeruginosa (Q1W548). Amino acids that occur in all bacteriocins are shaded red, and those that occur in most but not all strains are shaded yellow. B, localization of the conserved amino acids in the colicin M structure. C, distribution of surface-exposed positively charged (blue) and negatively charged (red) amino acids.α1–α9,α-helices;β1–β8,β-strands; andη1–η6, interhelical regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2533080&req=5

fig4: A, comparison of the colicin M amino acid sequence with the sequences of predicted bacteriocins of Burkholderia amifaria MC40-6 (accession number AOTM72), Burkholderia cepacia (QOBIY7), Pseudomonas syringae pv. tomato (Q88A25), and Pseudomonas aeruginosa (Q1W548). Amino acids that occur in all bacteriocins are shaded red, and those that occur in most but not all strains are shaded yellow. B, localization of the conserved amino acids in the colicin M structure. C, distribution of surface-exposed positively charged (blue) and negatively charged (red) amino acids.α1–α9,α-helices;β1–β8,β-strands; andη1–η6, interhelical regions.

Mentions: Central Domain—The central domain formed by residues 36–140 is globular and entirely α-helical (Figs. 1 and 2). The domain consists of six helices (α1–α6) of different lengths; five of these helices are wrapped around the longest central helix, α3, formed between residues Pro-70 and Leu-92. Residues Leu-36 to Gln-46 of α1 (Fig. 2A and Fig. 4A, LLVQVVYSFFQ) are strongly hydrophobic and extend outward from the globular domain. It is particularly appealing to hypothesize that this helix attaches colicin M to the cytoplasmic membrane in close proximity to the substrate, undecaprenyl-PP-MurNAc(pentapeptide)-GlcNAc, or binds to a portion of the C55 polyisoprenoid. A functional importance of this helix is supported by its conservation among homologous bacteriocins of colicin M (Fig. 4A). In the predicted receptor binding and translocation domains, only this region is somewhat conserved. α3 and α6 form the hydrophobic core of the central domain, which displays the lowest B-factors within the entire protein (Fig. 1B). Although there are no strong ionic interactions discernible between the central domain and the C-domain (Fig. 2C), a small β-sheet (β1, β2, β6, and β7) connects the two domains by hydrogen bridges and may contribute to the overall stability of the protein. A mutant in this region, R115C, is defective in colicin M uptake (6).


Crystal structure of colicin M, a novel phosphatase specifically imported by Escherichia coli.

Zeth K, Römer C, Patzer SI, Braun V - J. Biol. Chem. (2008)

A, comparison of the colicin M amino acid sequence with the sequences of predicted bacteriocins of Burkholderia amifaria MC40-6 (accession number AOTM72), Burkholderia cepacia (QOBIY7), Pseudomonas syringae pv. tomato (Q88A25), and Pseudomonas aeruginosa (Q1W548). Amino acids that occur in all bacteriocins are shaded red, and those that occur in most but not all strains are shaded yellow. B, localization of the conserved amino acids in the colicin M structure. C, distribution of surface-exposed positively charged (blue) and negatively charged (red) amino acids.α1–α9,α-helices;β1–β8,β-strands; andη1–η6, interhelical regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533080&req=5

fig4: A, comparison of the colicin M amino acid sequence with the sequences of predicted bacteriocins of Burkholderia amifaria MC40-6 (accession number AOTM72), Burkholderia cepacia (QOBIY7), Pseudomonas syringae pv. tomato (Q88A25), and Pseudomonas aeruginosa (Q1W548). Amino acids that occur in all bacteriocins are shaded red, and those that occur in most but not all strains are shaded yellow. B, localization of the conserved amino acids in the colicin M structure. C, distribution of surface-exposed positively charged (blue) and negatively charged (red) amino acids.α1–α9,α-helices;β1–β8,β-strands; andη1–η6, interhelical regions.
Mentions: Central Domain—The central domain formed by residues 36–140 is globular and entirely α-helical (Figs. 1 and 2). The domain consists of six helices (α1–α6) of different lengths; five of these helices are wrapped around the longest central helix, α3, formed between residues Pro-70 and Leu-92. Residues Leu-36 to Gln-46 of α1 (Fig. 2A and Fig. 4A, LLVQVVYSFFQ) are strongly hydrophobic and extend outward from the globular domain. It is particularly appealing to hypothesize that this helix attaches colicin M to the cytoplasmic membrane in close proximity to the substrate, undecaprenyl-PP-MurNAc(pentapeptide)-GlcNAc, or binds to a portion of the C55 polyisoprenoid. A functional importance of this helix is supported by its conservation among homologous bacteriocins of colicin M (Fig. 4A). In the predicted receptor binding and translocation domains, only this region is somewhat conserved. α3 and α6 form the hydrophobic core of the central domain, which displays the lowest B-factors within the entire protein (Fig. 1B). Although there are no strong ionic interactions discernible between the central domain and the C-domain (Fig. 2C), a small β-sheet (β1, β2, β6, and β7) connects the two domains by hydrogen bridges and may contribute to the overall stability of the protein. A mutant in this region, R115C, is defective in colicin M uptake (6).

Bottom Line: The novel phosphatase domain displays no sequence similarity to known phosphatases.The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins.The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, Tübingen, Germany.

ABSTRACT
Colicins are cytotoxic proteins secreted by certain strains of Escherichia coli. Colicin M is unique among these toxins in that it acts in the periplasm and specifically inhibits murein biosynthesis by hydrolyzing the pyrophosphate linkage between bactoprenol and the murein precursor. We crystallized colicin M and determined the structure at 1.7A resolution using x-ray crystallography. The protein has a novel structure composed of three domains with distinct functions. The N-domain is a short random coil and contains the exposed TonB box. The central domain includes a hydrophobic alpha-helix and binds presumably to the FhuA receptor. The C-domain is composed of a mixed alpha/beta-fold and forms the phosphatase. The architectures of the individual modules show no similarity to known structures. Amino acid replacements in previously isolated inactive colicin M mutants are located in the phosphatase domain, which contains a number of surface-exposed residues conserved in predicted bacteriocins of other bacteria. The novel phosphatase domain displays no sequence similarity to known phosphatases. The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins. The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains.

Show MeSH
Related in: MedlinePlus