title: Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase.
abstract: 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.
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy.
authors: Castrese
authors: C
authors: Morrone
authors: DiSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale 'A. Avogadro', Via Bovio 6, 28100 Novara, Italy.
authors: Riccardo
authors: R
authors: Miggiano
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy.
authors: Mario
authors: M
authors: Serpe
authors: DiSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale 'A. Avogadro', Via Bovio 6, 28100 Novara, Italy.
authors: Alberto
authors: A
authors: Massarotti
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy.
authors: Anna
authors: A
authors: Valenti
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy.
authors: Giovanni
authors: G
authors: Del Monaco
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy.
authors: Mosè
authors: M
authors: Rossi
authors: DiSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale 'A. Avogadro', Via Bovio 6, 28100 Novara, Italy.
authors: Franca
authors: F
authors: Rossi
authors: DiSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale 'A. Avogadro', Via Bovio 6, 28100 Novara, Italy.
authors: Menico
authors: M
authors: Rizzi
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy. Electronic address: giuseppe.perugino@ibbr.cnr.it.
authors: Giuseppe
authors: G
authors: Perugino
authors: Institute of Biosciences and BioResources, National Research Council of Italy, Via P. Castellino 111, 80125 Naples, Italy. Electronic address: maria.ciaramella@ibbr.cnr.it.
authors: Maria
authors: M
authors: Ciaramella
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000477:Alkylating Agents / Q000378:metabolism; D000478:Alkylation / Q000502:physiology; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000072417:Protein Domains; D055550:Protein Stability; D048229:Sulfolobus solfataricus / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D004268:DNA-Binding Proteins; D004247:DNA; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase
keywords: Alkylation damage; Conformational changes; Crystal structure; DNA repair; Fluorescent-based assay