The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins.
NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.
Oberer
Monika
M
Institut für Chemie, Arbeitsgruppe Strukturbiologie, Karl-Franzens-Universität Graz, Heinrichstrasse 28, A-8010 Graz, Austria.
Zangger
Klaus
K
Prytulla
Stefan
S
Keller
Walter
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D027101:DNA Topoisomerase IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014270:Trifluoroethanol / Q000494:pharmacology
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C489748:ParD protein, Plasmid RK2; C078613:parD protein, Bacteria; D014270:Trifluoroethanol; D027101:DNA Topoisomerase IV