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Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation - PubMed

  • ️Wed Jan 01 2003

Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation

Anton Meinhart et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from Streptococcus pyogenes carries the genes encoding the antitoxin/toxin system epsilon/zeta and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic epsilon(2)zeta(2) protein complex at a 1.95-A resolution and site-directed mutagenesis showed that free zeta acts as phosphotransferase by using ATPGTP. In epsilon(2)zeta(2), the toxin zeta is inactivated because the N-terminal helix of the antitoxin epsilon blocks the ATPGTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized.

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Figures

Figure 1
Figure 1

(a) Structure of the ɛ2ζ2 heterotetramer. The green line near the center shows the NCS axis. α-Helices of proteins ɛ and ζ are yellow and red, respectively, and β-strands of protein ζ are green. (b) Heterodimeric ɛ,ζ-half in stereo; coloring as in a and labeling of secondary structure elements as in c. The well ordered water molecules in the ATP site (the large crevice near the center of the complex) and the substrate site (closer to the viewer and located between α-helices E and F) are indicated by magenta spheres. (c) Topography of secondary structure elements; α-helices are shown as circles labeled in lower- and uppercase letters in proteins ɛ and ζ, respectively, and β-strands in ζ as triangles and numbered. Black numbers give the respective N- and C-terminal residues within the amino acid sequences.

Figure 2
Figure 2

Superposition of the ATP-binding site of Cmp with the large crevice in protein ζ. α-Helices of the latter are red, β-strands are green, relevant side chains are yellow and labeled, and side chains in Cmp are gray and not labeled. (a) The open conformation of Cmp with inorganic phosphate (gray; sulfate ion in protein ζ are yellow/red) bound to the P-loop (Lys-46ζ in protein ζ). (b) Superposition as in a, but both enzymes with bound ATP and of Mg2+ according to the Cmp crystal structure; the binding site for the yet unknown substrate of protein ζ is indicated. Conformations of Arg-158ζ and Arg-171ζ (with the appending loop region) are modeled according to the analogous amino acids in the closed conformation of Cmp and in NMP kinases.

Figure 3
Figure 3

Cut through protein ζ (stereo view) showing the substrate-binding site (right) forming a hydrophilic entrance to the large crevice of ζ to which ATP is modeled as in Fig. 2b. Positive electrostatic potential is blue and negative red; maximum values are ±10 kT/e. Catalytically active amino acids and Mg2+ (yellow) are labeled. The two Arg and Mg2+ stabilize the negative charge of ATP, Glu-116ζ stabilizes Mg2+, and Asp-67ζ is essential for deprotonation of the substrate (not shown) that creates the functionally essential nucleophile.

Figure 4
Figure 4

Steric hindrance of ATP binding to ɛ2ζ2. The putative position of ATP is modeled according to Cmp and NMP kinases. α-Helices οf proteins ɛ and ζ are yellow and red, respectively, and β-strands are green and labeled as in Fig. 1c. The hydrogen bonds between Arg-158ζ and Glu-16ɛ are indicated by dashed lines. α-Helix a of ɛ covers the large crevice in ζ, its side chains filling the crevice and interfering with ATP binding. The substrate binding pocket is located at the bottom of the crevice.

Figure 5
Figure 5

Sequence alignment of gene products from Enterococci, Lactococci, and Streptococci that are related to proteins ɛ and ζ. Secondary structure elements of proteins ɛ and ζ are indicated by cylinders (helices) and arrows (β-strands) on top of the alignments. Coloring and labeling are as in Fig. 1 b and c. Amino acids of protein ɛ that are shown in Fig. 4 to interfere with binding of ATP to the ɛ2ζ2 complex and catalytically important amino acids in protein ζ are red. Parts of the amino acid sequence that are conserved in four or more sequences are beige, and those conserved in only three sequences are blue.

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