The structural mechanism of the inhibition of archaeal RelE toxin by its cognate RelB antitoxin.
The archaeal toxin, aRelE, in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 inhibits protein synthesis, whereas its cognate antitoxin, aRelB, neutralizes aRelE activity by forming a non-toxic complex, aRelB-aRelE. The structural mechanism whereby aRelB neutralizes aRelE activity was examined by biochemical and biophysical analyses. Overexpression of aRelB with an aRelE mutant (ΔC6), in which the C-terminal residues critical for aRelE activity were deleted, in Escherichia coli allowed a stable complex, aRelB-ΔC6, to be purified. Isothermal titration of aRelE or ΔC6 with aRelB indicated that the association constant (Ka) of wild-type aRelB-aRelE is similar to that of aRelB-ΔC6, demonstrating that aRelB makes little contact with the C-terminal active site of aRelE. Overexpression of deletion mutants of aRelB with aRelE indicated that either the N-terminal (pos. 1-27) or C-terminal (pos. 50-67) fragment of aRelB is sufficient to counteract the toxicity of aRelE in E. coli cells and the second α-helix (α2) in aRelB plays a critical role in forming a stable complex with aRelE. The present results demonstrate that aRelB, as expected from its X-ray structure, precludes aRelE from entering the ribosome, wrapping around the molecular surface of aRelE.
Shinohara
Masaaki
M
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
Guo
Jin Xu
JX
Mori
Misako
M
Nakashima
Takashi
T
Takagi
Hisanori
H
Nishimoto
Etsuko
E
Yamashita
Shoji
S
Tsumoto
Kouhei
K
Kakuta
Yoshimitsu
Y
Kimura
Makoto
M
D000992:Antitoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D017353:Gene Deletion; D043902:Pyrococcus horikoshii / Q000235:genetics / Q000378:metabolism*; D014118:Toxins, Biological / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics
D000992:Antitoxins; D019843:Archaeal Proteins; D014118:Toxins, Biological