The bacterial toxin ColE7 bears an HNH motif which has been identified in hundreds of prokaryotic and eukaryotic endonucleases, involved in DNA homing, restriction, repair, or chromosome degradation. The crystal structure of the nuclease domain of ColE7 in complex with a duplex DNA has been determined at 2.5 A resolution. The HNH motif is bound at the minor groove primarily to DNA phosphate groups at and beyond the 3' side of the scissile phosphate, with little interaction with ribose groups and bases. This result provides a structural basis for sugar- and sequence-independent DNA recognition and the inhibition mechanism by inhibitor Im7, which blocks the substrate binding site but not the active site. Structural comparison shows that two families of endonucleases bind and bend DNA in a similar way to that of the HNH ColE7, indicating that endonucleases containing a "betabetaalpha-metal" fold of active site possess a universal mode for protein-DNA interactions.

The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-A crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel ci-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin.E-group colicins (from El to E9) are plasmid-borne antibioticlike bacteriocins that are active against sensitive Escherichia coli and closely related coliform bacteria (1). They bind to the vitamin B12 receptor, BtuB, and subsequently translocate across the outer and cytoplasmic membranes, inducing cell death (2). Three cytotoxic classes of E-group colicins have thus far been identified, including pore-forming colicins such as colicin El (3), RNase colicins such as colicins E3 (4) and E6 (5), and DNase colicins such as colicins E2 (6), E7 (7), E8 (8), and E9 (7). Colicin E7 (ColE7) is a nonspecific endonuclease that causes both single-and double-stranded breaks in the DNA of sensitive cells. Production of ColE7 is regulated by a "SOS" response operon that encodes ColE7, ImmE7 (immunity protein of colicin E7), and a lysis protein for transportation of the ColE7/ImmE7 complex. Immediately after production, colicin forms a complex with its coordinately produced ImmE and thus neutralizes its toxicity toward the host cell. The DNase-type colicins contain almost identical sequences in their translocation and receptor recognition domains, which are located in the N-terminal two-thirds of the sequence.C-terminal endonuclease domains (T2A domain) of DNasetype colicins are =80% identical, and sequences of their corresponding immunity proteins are 60-70% identical (7).However, despite the high sequence identities in either colicins or immunity proteins, an immunity protein can only completely protect a cell from the action of its own cognate colicin (9). Neither the mechanism for the specific protein-protein interaction between colicins and immunity proteins nor the inhibition of toxicity incurred after the formation of colicin/ ImmE complex has been explained yet. Thus the crystal structure of ImmE7 may provide invaluable information, expanding our limited knowledge of the specific interactions between proteins. Here we report the 1.8-A crystal structure of the ImmE7 protein...

H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nucleaseColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn 2+ -bound and Mn 2+ -bound complexes in the presence of phosphate ions at resolutions of 2.6 Å to 2.0 Å. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel ␤-strands linked to a C-terminal ␣-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg 2+-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn 2+ or Mn 2+ ions were observed in the center of the H-N-H motif in cases of Zn 2+ or Mn 2+ -soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.

Site-specific cleavage of mRNA has been identified in vivo for the polycistronic colicin E7 operon (ColE7), which occurs between G and A nucleotides located at the Asp52 codon (GAT) of the immunity gene (ceiE7). In vitro, this specific cleavage occurs only in the presence of the ceiE7 gene product (ImmE7). The crystal structure of dimeric ImmE7 has been determined at 1.8 A resolution by X-ray crystallographic analysis. We found that several residues located at the interface of dimeric ImmE7 bear surprising resemblance to the active sites of some RNases. These results suggest that dimeric ImmE7 may possess a novel RNase activity that cleaves its own mRNA at a specific site and thus autoregulates translational expression of the downstream celE7 gene as well as degradation of the upstream ceaE7 mRNA.