National Center for
5LLQ: Crystal Structure Of Sulfolobus Solfataricus O6-methylguanine Methyltransferase C119f Variant
Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase
Biochim. Biophys. Acta (2016) 1861 p.86-96
BACKGROUND: Alkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. METHODS: As a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. RESULTS: Conformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. CONCLUSIONS: Our data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. GENERAL SIGNIFICANCE: A general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.