Recognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment. Structure, thermodynamics of binding, stability, and influence on interactions with DNA.

TitleRecognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment. Structure, thermodynamics of binding, stability, and influence on interactions with DNA.
Publication TypeJournal Article
Year of Publication2003
AuthorsLah, J., I. Marianovsky, G. Glaser, H. Engelberg-Kulka, J. Kinne, L. Wyns, and R. Loris
JournalJ Biol Chem
Volume278
Issue16
Pagination14101-11
Date Published2003 Apr 18
ISSN0021-9258
KeywordsAmino Acid Sequence, Animals, Binding Sites, Biochemistry, Calorimetry, Calorimetry, Differential Scanning, Camels, Circular Dichroism, Dimerization, DNA, DNA-Binding Proteins, Entropy, Escherichia coli, Escherichia coli Proteins, Immunoglobulin Fragments, Models, Molecular, Molecular Sequence Data, Peptides, Promoter Regions, Genetic, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Temperature, Thermodynamics, Time Factors
Abstract

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.

DOI10.1074/jbc.M209855200
Alternate JournalJ. Biol. Chem.
PubMed ID12533537