title: Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.
abstract: Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.
authors.affiliation: DSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, 28100 Novara, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Miggiano
authors.affiliation: Institute of Biosciences and Bioresources, IBBR-CNR, 80125 Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Perugino
authors.affiliation: Institute of Biosciences and Bioresources, IBBR-CNR, 80125 Naples, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Ciaramella
authors.affiliation: Institute of Biosciences and Bioresources, IBBR-CNR, 80125 Naples, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Serpe
authors.affiliation: IOCB-Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences v.v.i., 166 10 Prague 6, Czech Republic.
authors.forename: Dominik
authors.initials: D
authors.lastname: Rejman
authors.affiliation: IOCB-Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences v.v.i., 166 10 Prague 6, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Páv
authors.affiliation: IOCB-Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences v.v.i., 166 10 Prague 6, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Pohl
authors.affiliation: DSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, 28100 Novara, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Garavaglia
authors.affiliation: DSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, 28100 Novara, Italy.
authors.forename: Samarpita
authors.initials: S
authors.lastname: Lahiri
authors.affiliation: DSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, 28100 Novara, Italy menico.rizzi@uniupo.it franca.rossi@uniupo.it.
authors.forename: Menico
authors.initials: M
authors.lastname: Rizzi
authors.affiliation: DSF-Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, 28100 Novara, Italy menico.rizzi@uniupo.it franca.rossi@uniupo.it.
authors.forename: Franca
authors.initials: F
authors.lastname: Rossi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D003461:Crystallography; D004249:DNA Damage / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: DNA repair; DNA-binding protein; Mycobacterium tuberculosis; O6-methylguanine-DNA methyltransferase; co-operativity; crystal structure