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

Abstract

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...

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

“…2 Effect of Ligand Binding and Disulfide Bond Formation on the Ability of the E9 DNase to Interact with Lipids-The experiments described above were performed on protein preparations of the E9 DNase in the absence of bound metal. However, it has been shown previously that binding of a stoichiometric amount of Zn 2ϩ causes a considerable increase in the conformational stability of the E9 DNase (19). This is manifest by an increase of 22°C (from 37 to 59°C) in the melting temperature of the protein at pH 7.5, a considerable decrease in the susceptibility of the protein to proteolysis, and a decrease in affinity of 2 the protein for the hydrophobic dye ANS (19).…”

“…The protein was then dialysed against 3 ϫ 5 liters of 50 mM Tris-HCl, pH 7.5 containing 500 mM NaCl, 1 ϫ 5 liters of 50 mM Tris-HCl, pH 7.5 containing 200 mM NaCl, 5 liters of 50 mM Tris-HCl, pH 7.5, and 3 ϫ 5 liters of dH 2 O. The protein was verified as being free of contamination by both metal and EDTA by its ability to bind a stoichiometric amount of zinc as determined by ANS binding, as described by Pommer et al (19). The protein was then aliquoted, lyophilized, and stored at Ϫ20°C.…”

“…The intrinsic fluorescence of the E9 DNase is very sensitive to ligand-binding events, to the extent that binding at the immunity protein exosite and active site can be distinguished (12,19). Complex formation with immunity protein at the exosite gives rise to an enhancement in its intrinsic fluorescence, whereas binding of either transition metals or DNA at the active site of the protein quench the intrinsic fluorescence.…”

“…The protein was then aliquoted, lyophilized, and stored at Ϫ20°C. The concentration of the E9 DNase was determined from the absorbance at 280 nm using a molar extinction coefficient of 17,550 M Ϫ1 cm Ϫ1 (19). For experiments in which the zinc-bound protein was used, ZnCl 2 was added to give a Zn 2ϩ :E9 DNase ratio of 1.2:1.…”

“…The HNH motif is also found in a variety of endonucleases, including the caspase-activated DNase that is responsible for degradation of the chromosome during eukaryotic apoptosis (16 -18). The E9 DNase binds Zn 2ϩ ions with nM affinity, and this interaction considerably stabilizes the protein (19). However, for the DNase domain to be enzymatically active in vivo, Mg 2ϩ ions are required, although these do not bind directly to the protein in the absence of DNA (16,20).…”