title: Mapping the tRNA binding site on the surface of human DNMT2 methyltransferase.
abstract: The DNMT2 enzyme methylates tRNA-Asp at position C38. Because there is no tRNA-Dnmt2 cocrystal structure available, we have mapped the tRNA binding site of DNMT2 by systematically mutating surface-exposed lysine and arginine residues to alanine and studying the tRNA methylation activity and binding of the corresponding variants. After mutating 20 lysine and arginine residues, we identified eight of them that caused large (>4-fold) decreases in catalytic activity. These residues cluster within and next to a surface cleft in the protein, which is large enough to accommodate the tRNA anticodon loop and stem. This cleft is located next to the binding pocket for the cofactor S-adenosyl-L-methionine, and the catalytic residues of DNMT2 are positioned at its walls or bottom. Many of the variants with strongly reduced catalytic activity showed only a weak loss of tRNA binding or even bound better to tRNA than wild-type DNMT2, which suggests that the enzyme induces some conformational changes in the tRNA in the transition state of the methyl group transfer reaction. Manual placement of tRNA into the structure suggests that DNMT2 mainly interacts with the anticodon stem and loop.
authors.affiliation: Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Tomasz P
authors.initials: TP
authors.lastname: Jurkowski
authors.forename: Raghuvaran
authors.initials: R
authors.lastname: Shanmugam
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D012343:RNA, Transfer; D004248:DNA (Cytosine-5-)-Methyltransferases; C112218:TRDMT1 protein, human