O6-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 -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.