The archaeal toxin, aRelE, in the hyperthermophilic archaeon Pyrococcus horikoshu 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 (Delta C6), in which the C-terminal residues critical for aRelE activity were deleted, in Escherichia coli allowed a stable complex, aRelB-Delta C6, to be purified Isothermal titration of aRelE or Delta C6 with aRelB indicated that the association constant (Ka) of wild-type aRelB-aRelE is similar to that of aRelB-Delta C6, 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 alpha-helix (alpha 2) 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 (C) 2010 Elsevier Inc. All rights reserved