title: Thermodynamic consequences of bipartite immunity protein binding to the ribosomal ribonuclease colicin E3.
abstract: Colicin E3 is a 60 kDa, multidomain protein antibiotic that targets its ribonuclease activity to an essential region of the 16S ribosomal RNA of Escherichia coli. To prevent suicide of the producing cell, synthesis of the toxin is accompanied by the production of a 10 kDa immunity protein (Im3) that binds strongly to the toxin and abolishes its enzymatic activity. In the present work, we study the interaction of Im3 with the isolated cytotoxic domain (E3 rRNase) and intact colicin E3 through presteady-state kinetics and thermodynamic measurements. The isolated E3 rRNase domain forms a high affinity complex with Im3 (K(d) = 10(-12) M, in 200 mM NaCl at pH 7.0 and 25 degrees C). The interaction of Im3 with full-length colicin E3 under the same conditions is however significantly stronger (K(d) = 10(-14) M). The difference in affinity arises almost wholly from a marked decrease in the dissociation rate constant for the full-length complex (8 x 10(-7) s(-1)) relative to the E3 rRNase-Im3 complex (1 x 10(-4) s(-1)), with their association rates comparable ( approximately 10(8) M(-1) s(-1)). Thermodynamic measurements show that complex formation is largely enthalpy driven. In light of the recently published crystal structure of the colicin E3-Im3 complex, the additional stabilization of the wild-type complex can be ascribed to the interaction of Im3 with the N-terminal translocation domain of the toxin. These observations suggest a mechanism whereby dissociation of the immunity protein prior to translocation into the target cell is facilitated by the loss of the Im3-translocation domain interaction.
authors.affiliation: Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Walker
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002151:Calorimetry; D003087:Colicins / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D003087:Colicins