Mechanism to Trigger Unfolding in O super(6)-Alkylguanine-DNA Alkyltransferase

• Published in: Chembiochem : a European journal of chemical biology Vol. 14; no. 6; pp. 703 - 710
• Main Authors: Brunk, Elizabeth, Mollwitz, Birgit, Rothlisberger, Ursula
• Format: Journal Article
• Language: English
• Published: 01-04-2013
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201200566

O super(6)-alkylguanine-DNA alkyltransferase (AGT) adopts a non-enzymatic suicide mechanism for the repair of methylated guanine bases by transferring the methyl adduct to itself, thereby initiating unfolding and fast degradation. Classical molecular dynamics simulations provide quantitative evidence that two conserved glycine residues at the centre of an [alpha]-helix make the structure susceptible to structural perturbations. The stability of this helix, designated the "recognition helix", is an important factor during the early onset of unfolding of human AGT (hAGT). By combining theory and experiment, we found that helical stability is controlled by key factors in the surrounding protein structure. By using a "double-clip" mechanism, nearby residues hydrogen bond to both the base and centre of the helix. This double clip stabilises this site in the protein in the absence of substrate, but the helix is destabilised upon alkylation. The present investigation aimed to establish why alkylation of hAGT leads to conformational changes and how the protein environment functions as a switch, thus turning the stability of the protein "on" or "off" to tune degradability. Double trouble: In hAGT, a "double-clip" mechanism is used to stabilize the protein in the absence of alkylguanine and to trigger rapid unfolding upon alkyl transfer by creating steric repulsions between S159, M134 and alkylated C145.