title: 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.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
authors.forename: A J
authors.initials: AJ
authors.lastname: Pommer
authors.forename: U C
authors.initials: UC
authors.lastname: Kühlmann
authors.forename: A
authors.initials: A
authors.lastname: Cooper
authors.forename: A M
authors.initials: AM
authors.lastname: Hemmings
authors.forename: G R
authors.initials: GR
authors.lastname: Moore
authors.forename: R
authors.initials: R
authors.lastname: James
authors.forename: C
authors.initials: C
authors.lastname: Kleanthous
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D003035:Cobalt / Q000378:metabolism; D003087:Colicins; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D008670:Metals; C007104:colicin immunity proteins; C488859:immE9 protein, E coli; D003035:Cobalt; D009532:Nickel; D003851:Deoxyribonucleases; D008274:Magnesium; D015032:Zinc