Characterization of Dual Substrate Binding Sites in the Homodimeric Structure of Escherichia coli mRNA Interferase MazF

MazF and MazE constitute a so-called addiction module that is critical for bacterial growth arrest and eventual cell death in response to stress. The MazF toxin was recently shown to possess mRNA interferase (MIase) activity, and acts as a protein synthesis inhibitor by cleaving cellular mRNA. As a cognate regulator, the short-lived antitoxin, MazE, inhibits MazF MIase activity and hence maintains the delicate homeostasis between these two components. In the present study, we have shown that the MazF homodimer contains two symmetric binding sites, each of which is capable of interacting with a MazE C-terminal peptide, MazEp(54–77). The slow exchange phenomenon between free and peptide-bound MazF on the NMR timescale indicates relatively high affinities for MazEp(54–77) at both sites (Kd,K′d<10−7 M). However, the observed sequential binding behavior suggests a negative cooperativity between the two sites (Kd