Non-cytotoxic variants of the Kid protein that retain their auto-regulatory activity.
Kid and Kis are, respectively, the toxin and antitoxin encoded by the parD operon of plasmid R1. The recently solved crystal structure of Kid has revealed that this protein closely resembles the CcdB toxin of plasmid F. In CcdB, the residues involved in toxicity are located at the carboxy-terminal end of the protein. However, an analogous information on the Kid toxin was not available. Here, we have characterized a collection of non-toxic mutants of the Kid protein and identified the residues that affected the toxicity but not the co-regulatory activity of Kid. These are located in two discrete regions of the protein, at the amino and carboxy-terminal ends. Particularly, residues E18 and R85, that are conserved in the Escherichia coli ChpAK and RelE toxins, are affected by amino-acid changes that alter neither the overall structure of the protein nor its state of association, as shown by CD and sedimentation equilibrium analyses. However, thermal denaturation and intrinsic tryptophan fluorescence emission data point to subtle local changes at the N-terminal end of the protein. The implications of these results in the current model on the structure and function of Kid-related bacterial toxins are discussed.
Santos-Sierra
Sandra
S
Departemento de Microbiologia Molecular, Centro de Investigaciones Biológicas, CSIC, Velázquez 144, Madrid E-28006, Spain.
Lemonnier
Marc
M
Nuñez
Belen
B
Hargreaves
David
D
Rafferty
John
J
Giraldo
Rafael
R
Andreu
Jose Manuel
JM
Díaz-Orejas
Ramon
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D002874:Chromosome Mapping; D003603:Cytotoxins / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D005453:Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D020836:Protein Structure, Quaternary; D013057:Spectrum Analysis; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D017931:DNA Primers; C068353:Kid protein, Bacteria