title: 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.
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.
authors.affiliation: Department of Ultrastructure, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 St. Genesius Rode, Belgium. jurij.lah@uni-lj.si
authors.forename: Jurij
authors.initials: J
authors.lastname: Lah
authors.forename: Irina
authors.initials: I
authors.lastname: Marianovsky
authors.forename: Gad
authors.initials: G
authors.lastname: Glaser
authors.forename: Hanna
authors.initials: H
authors.lastname: Engelberg-Kulka
authors.forename: Jörg
authors.initials: J
authors.lastname: Kinne
authors.forename: Lode
authors.initials: L
authors.lastname: Wyns
authors.forename: Remy
authors.initials: R
authors.lastname: Loris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002162:Camelus; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D019277:Entropy; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; C422954:MazE protein, E coli; D010455:Peptides; D004247:DNA