Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 2; pp. 86 - 96
• Main Authors: Morrone, Castrese, Miggiano, Riccardo, Serpe, Mario, Massarotti, Alberto, Valenti, Anna, del Monaco, Giovanni, Rossi, Mosè, Rossi, Franca, Rizzi, Menico, Perugino, Giuseppe, Ciaramella, Maria
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2017
• Subjects: active sites; Alkyl and Aryl Transferases - metabolism; Alkylating Agents - metabolism; alkylation; Alkylation - physiology; Alkylation damage; Catalysis; catalytic activity; Conformational changes; Crystal structure; cysteine; DNA; DNA - metabolism; DNA damage; DNA repair; DNA Repair - physiology; DNA-Binding Proteins - metabolism; drug therapy; Fluorescent-based assay; molecular dynamics; Protein Domains; Protein Stability; proteins; Sulfolobus solfataricus; Sulfolobus solfataricus - metabolism; thermal stability; Alkylation damage; RMSD; SsOGTm; DNA repair; Conformational changes; Fluorescent-based assay; O6-MG; DSF; hAGT; Nter; MD; O6-BG; BG-VG; SsOGT; DDMP; dsDNAm; Cter; Crystal structure
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.10.020

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. 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. 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. 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. 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. [Display omitted] •The thermostable DNA repair SsOGT is a suitable model for structural and biochemical studies of AGTs.•Interdomain locks safeguard folding and activity of SsOGT.•Step-by-step conformational modifications of SsOGT are associated with lesion recognition and DNA repair.