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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.

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Similarities of colicin M to other protein structures. A, simulated annealed 2Fo - Fc map of the N-terminal TonB-box domain. B, superposition of the N termini of colicin M (white), FhuA (magenta; in complex with a TonB fragment; PDB entry 2GRX), and BtuB (cyan; in complex with a TonB fragment; PDB entry 2GSK). C, superposition of the C-terminal domain of colicin M (gray) with the membrane-spanning part of the outer membrane transporter Hia of H. influenzae (red, blue, and green) (39).
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fig3: Similarities of colicin M to other protein structures. A, simulated annealed 2Fo - Fc map of the N-terminal TonB-box domain. B, superposition of the N termini of colicin M (white), FhuA (magenta; in complex with a TonB fragment; PDB entry 2GRX), and BtuB (cyan; in complex with a TonB fragment; PDB entry 2GSK). C, superposition of the C-terminal domain of colicin M (gray) with the membrane-spanning part of the outer membrane transporter Hia of H. influenzae (red, blue, and green) (39).

Mentions: The TonB box (Fig. 2B, residues 2–8) is exposed on the surface and thus may be accessible to interact with TonB. This N-terminal arrangement is stabilized only by a small number of weak interactions, mainly H-bonds to the backbone of the following central domain, and may be detached to interact with TonB. Genetic data support the involvement of the TonB box in interaction with TonB. Mutations in the TonB box (L4N, L4P, V6R, V6G, and V6E) inactivate colicin M. Sensitivity to colicin M is restored in cells in which the mutation V6R is combined with the chromosomal TonB mutation Q160L. Suppression of the V6R mutation by the Q160L mutation suggests interaction of colicin M with TonB through regions in which these mutations are localized (6). In FhuA, the TonB box is not visible (32, 33), but co-crystals of FhuA with a C-proximal fragment of TonB (residues 158–235) reveal a FhuA N-proximal β-strand (TonB box) bound to a three-stranded β-sheet of TonB (8). A similar structure of a TonB fragment bound to the BtuB vitamin B12 transporter has been determined (34). In colicin M, the TonB box displays an elongated and well structured motif (Fig. 3A) that can be overlaid onto the structures of FhuA and BtuB bound to the TonB fragment (Fig. 3B). In colicin B, the TonB box is folded back and attached to the N-terminal lobe (31). The TonB box of colicin Ia interacts along one surface of the N-proximal helical sheet (26).


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)

Similarities of colicin M to other protein structures. A, simulated annealed 2Fo - Fc map of the N-terminal TonB-box domain. B, superposition of the N termini of colicin M (white), FhuA (magenta; in complex with a TonB fragment; PDB entry 2GRX), and BtuB (cyan; in complex with a TonB fragment; PDB entry 2GSK). C, superposition of the C-terminal domain of colicin M (gray) with the membrane-spanning part of the outer membrane transporter Hia of H. influenzae (red, blue, and green) (39).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Similarities of colicin M to other protein structures. A, simulated annealed 2Fo - Fc map of the N-terminal TonB-box domain. B, superposition of the N termini of colicin M (white), FhuA (magenta; in complex with a TonB fragment; PDB entry 2GRX), and BtuB (cyan; in complex with a TonB fragment; PDB entry 2GSK). C, superposition of the C-terminal domain of colicin M (gray) with the membrane-spanning part of the outer membrane transporter Hia of H. influenzae (red, blue, and green) (39).
Mentions: The TonB box (Fig. 2B, residues 2–8) is exposed on the surface and thus may be accessible to interact with TonB. This N-terminal arrangement is stabilized only by a small number of weak interactions, mainly H-bonds to the backbone of the following central domain, and may be detached to interact with TonB. Genetic data support the involvement of the TonB box in interaction with TonB. Mutations in the TonB box (L4N, L4P, V6R, V6G, and V6E) inactivate colicin M. Sensitivity to colicin M is restored in cells in which the mutation V6R is combined with the chromosomal TonB mutation Q160L. Suppression of the V6R mutation by the Q160L mutation suggests interaction of colicin M with TonB through regions in which these mutations are localized (6). In FhuA, the TonB box is not visible (32, 33), but co-crystals of FhuA with a C-proximal fragment of TonB (residues 158–235) reveal a FhuA N-proximal β-strand (TonB box) bound to a three-stranded β-sheet of TonB (8). A similar structure of a TonB fragment bound to the BtuB vitamin B12 transporter has been determined (34). In colicin M, the TonB box displays an elongated and well structured motif (Fig. 3A) that can be overlaid onto the structures of FhuA and BtuB bound to the TonB fragment (Fig. 3B). In colicin B, the TonB box is folded back and attached to the N-terminal lobe (31). The TonB box of colicin Ia interacts along one surface of the N-proximal helical sheet (26).

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