Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF

Guang Yao Li, Yonglong Zhang, Mitchell C.Y. Chan, Tapas Mal, Klaus P. Hoeflich, Masayori Inouye, Mitsuhiko Ikura

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

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<K′ d). A 13 base single-stranded DNA, employed as an uncleavable RNA substrate analog, can also bind to both sites on the MazF homodimer with moderate affinity (Kd∼10-5-10-6 M). Chemical shift perturbation data deduced from NMR experiments indicates that the two binding sites for the MazEp peptide coincided with those for the single-stranded DNA competitive inhibitor. These dual substrate-binding sites are located on the concave interface of the MazF homodimer, consisting of a highly basic region underneath the S1-S2 loop and two hydrophobic regions containing the H1 helix of one subunit and the S3-S4 loop of the opposing subunit. We show that the MazF homodimer is a bidentate endoribonuclease equipped with two identical binding sites for mRNA processing and that a single MazE molecule occupying one of the binding sites can affect the conformation of both sites, hence efficiently hindering the activity of MazF.

Original languageEnglish (US)
Pages (from-to)139-150
Number of pages12
JournalJournal of Molecular Biology
Volume357
Issue number1
DOIs
StatePublished - Mar 17 2006

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Binding Sites
Escherichia coli
Messenger RNA
Single-Stranded DNA
Peptides
Endoribonucleases
Antitoxins
Protein Synthesis Inhibitors
Homeostasis
Cell Death
RNA
Growth

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Cite this

Li, Guang Yao ; Zhang, Yonglong ; Chan, Mitchell C.Y. ; Mal, Tapas ; Hoeflich, Klaus P. ; Inouye, Masayori ; Ikura, Mitsuhiko. / Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF. In: Journal of Molecular Biology. 2006 ; Vol. 357, No. 1. pp. 139-150.
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abstract = "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<K′ d). A 13 base single-stranded DNA, employed as an uncleavable RNA substrate analog, can also bind to both sites on the MazF homodimer with moderate affinity (Kd∼10-5-10-6 M). Chemical shift perturbation data deduced from NMR experiments indicates that the two binding sites for the MazEp peptide coincided with those for the single-stranded DNA competitive inhibitor. These dual substrate-binding sites are located on the concave interface of the MazF homodimer, consisting of a highly basic region underneath the S1-S2 loop and two hydrophobic regions containing the H1 helix of one subunit and the S3-S4 loop of the opposing subunit. We show that the MazF homodimer is a bidentate endoribonuclease equipped with two identical binding sites for mRNA processing and that a single MazE molecule occupying one of the binding sites can affect the conformation of both sites, hence efficiently hindering the activity of MazF.",
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Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF. / Li, Guang Yao; Zhang, Yonglong; Chan, Mitchell C.Y.; Mal, Tapas; Hoeflich, Klaus P.; Inouye, Masayori; Ikura, Mitsuhiko.

In: Journal of Molecular Biology, Vol. 357, No. 1, 17.03.2006, p. 139-150.

Research output: Contribution to journalArticle

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