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The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn 2؉ or Ni 2؉ ) the role of which is unknown. In the present work we find that neither Zn 2؉ nor Ni 2؉ is required for DNase activity, which instead requires Mg 2؉ ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn 2؉ , Ni 2؉ , or Co 2؉ , the thermodynamic stability of the domain increased substantially, and that the equilibrium dissociation constant for Zn 2؉ was less than or equal to nanomolar, while that for Co 2؉ and Ni 2؉ was micromolar. Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.Colicins are a formidable weapon in the armory of a bacterium as it competes for nutrients with other bacteria, exemplified by the first-order kinetics of colicin-mediated cell death, which imply that a single toxin molecule is sufficient to kill a cell (1). Colicins have been intensively studied since the late 1950s (2), with much of this work revolving around how these folded proteins are able to traverse the membrane barriers of a bacterium (reviewed in Ref. 3). This process is distinct from that of mitochondrial import in eukaryotic cells since colicins do not possess signal sequences but rather are dependent on the activities of three domains: a central receptor recognition domain involved in binding to outer membrane nutrient receptors; an N-terminal translocation domain, which, through its interactions with several periplasmic proteins, causes the outer membrane to be breached allowing the C-terminal cytotoxic domain to enter the periplasm. This latter activity is most often a voltage-gated ionophore (colicins A, B, Ia, E1, and N, for example), which associates with the inner membrane as a molten globule, and then depolarizes the cell (4 -6). Cell death ensues through the efflux of potassium and other ions out of the cell.Of the many different types of colicin that have been identified, perhaps the most unusual are the nuclease family of E colicins (reviewed in Ref. 7). Nuclease colicins require the outer membrane vitamin B 12 receptor BtuB as well as the porin OmpF and the Tol proteins (located in both the periplasm and inner membrane) for import. This complex machinery is needed to translocate the cytotoxic domain across both membranes of Esche...

The crystal structure of the cytotoxic endonuclease domain from the bacterial toxin colicin E9 in complex with its cognate immunity protein Im9 reveals that the inhibitor does not bind at the active site, the core of which comprises the HNH motif found in intron-encoded homing endonucleases, but rather at an adjacent position leaving the active site exposed yet unable to bind DNA because of steric and electrostatic clashes with incoming substrate. Although its mode of action is unorthodox, Im9 is a remarkably effective inhibitor since it folds within milliseconds and then associates with its target endonuclease at the rate of diffusion to form an inactive complex with sub-femtomolar binding affinity. This hyperefficient mechanism of inhibition could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active site.

Homing endonucleases are classified into four families based on active site sequence motifs. Through structural comparisons we have found structural similarities between the endonuclease domain of colicin E9, an H-N-H motif-containing enzyme, and both the non-specific nuclease from Serratia and I-PpoI, a His-Cys box-containing homing endonuclease. Our comparison identifies conservation at the heart of all three enzyme active sites and so argues for a re-classification of H-N-H and His-Cys box homing endonucleases as a single family. We suggest the`L LL LK K-Me family' of homing enzymes to reflect the three elements of secondary structure and the metal ion that define the motif.z 1999 Federation of European Biochemical Societies.

The driving force in the progression of COPD is the development of exacerbations which are mostly the result of excessive inflammation. Bronchodilatators play an important role in the treatment of COPD. The reported reduction in exacerbation rates in COPD is due to the inhibition of vagal-mediated bronchoconstriction and mucus secretion. However, recent studies have highlighted the existence of muscarinic receptors on inflammatory cells and we have explored the possibility that tiotropium bromide might also inhibit neutrophil migration. We analysed the influence of tiotropium on the release of neutrophil chemotactic activity in response to acetylcholine (ACh) and the expression of muscarinic receptors on human alveolar macrophages (AM), A549 cells, MonoMac6 cells, and human lung fibroblasts. We found significant levels of all muscarinic receptor subtypes on all analysed cells except the fibroblasts. Fibroblasts expressed predominantly M2, receptors and did not release chemotactic activity. AM, A549 cells, and MonoMac6 cells released chemotactic active mediators after incubation with ACh. The secretion could be suppressed by more than 70% after coincubation with tiotropium. Tiotropium alone did not influence the granulocyte migration. Most of the chemotactic activity could be attributed to leukotriene B4 (LTB4). The release of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) was not induced by ACh. From this, we suggest that the suppression of the Ach-mediated release of chemotactic substances like LTB4 modulates the inflammatory reaction. This may contribute to the decreased rate of exacerbations in COPD, which was observed in clinical trials.

Our data support the hypothesis that DP8 and/or DP9 represent additional pharmacological targets for the suppression of T cell proliferation and for anti-inflammatory therapy.

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