The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface.

Abstract

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...

Measurement of histidine pKavalues and tautomer populations in invisible protein states

“…Notably, neither H40 nor H47 exhibit the 4:1 ratio in the native conformation of the protein, with H40 predominantly in the rare δ tautomer form and H47 exclusively in the « state, suggesting that both histidines are in hydrogen-bonded environments. Indeed, crystal structures of Im7 show that H47 stacks on W75 and is hydrogen bonded to the backbone amide of D49, whereas H40 becomes buried by the N terminus in the native state, potentially forming a hydrogen bond with the backbone carbonyl of K4 (77,78). Thus, significant rearrangements of the histidine side chains must accompany the I-to-N transition, leading to burial of the exposed H40 and H47 side chains.…”

The role of the N-terminal loop in the function of the colicin E7 nuclease domain

“…In order to exert cell killing activity, ColE7 has to get across both the outer and the inner cell membrane, facilitated by the receptor-binding and translocation domains [3,4]. The host cell itself is protected by the simultaneously expressed immunity protein Im7 blocking the DNA-binding site [5,6] of the NColE7 domain due to tight interactions based on charge-complementarity [7][8][9][10][11][12].…”

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

“…1). The E7 DNaseIm7 complex was expressed as a histidine-tagged complex (in which the Im7 protein carried the tag) and the structure solved by molecular replacement using the previously solved Im7 crystal structure (22,26). Atomic absorption experiments indicated that zinc was the predominant metal in this complex, although nickel could also be detected (22).…”