Abstract
Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.
Highlights
Bacterial populations are capable of overcoming periods of harsh conditions that are normally lethal for metabolically active cells through the stochastic generation of persisters [1, 2]
Loop 4-5 is observed in two distinct conformations in chains A and B of crystal form II, whereas the conformations observed in the remaining eight monomers correspond to an ensemble of closely related conformations
Molecular Framework for MazF Substrate Recognition and Catalysis—Knowledge of the structural basis of substrate recognition by MazF proteins has been limited to the crystal structure of a substrate mimic complex of Bacillus subtilis MazF (BsMazF), which shares only 24% sequence identity with the E. coli enzyme
Summary
Y is U, A, or G [23]. In addition, not all MazF recognition sites are cleaved in each mRNA, indicating that RNA secondary structure plays a major role in directing MazF activity [17, 23]. We describe the structures of wild type and E24A mutant of E. coli MazF in their free forms and in complex with a 7-nucleotide substrate mimic or a peptide corresponding to the 15 C-terminal residues of E. coli MazE, respectively. These structures together with activity and ITC data provide novel insights into substrate recognition and regulation of E. coli MazF and further support an evolutionary relationship and a common regulatory mechanism for ccdAB and mazEF
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
