title: Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism.
abstract: Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg(2+). A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg(2+) from Ca(2+). Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA.
authors.forename: Zihan
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA.
authors.forename: Anna Y
authors.initials: AY
authors.lastname: Lynn
authors.affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA.
authors.forename: Brittany
authors.initials: B
authors.lastname: Varnado
authors.affiliation: Molecular Targets Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Beutler
authors.affiliation: Department of Chemistry, Brooklyn College, City University of New York, Brooklyn, NY 11210, USA.
authors.forename: Ryan P
authors.initials: RP
authors.lastname: Murelli
authors.affiliation: Basic Research Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.affiliation: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA tangl@ku.edu.
authors.forename: Liang
authors.initials: L
authors.lastname: Tang
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D042003:DNA Packaging; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D017902:Podoviridae / Q000201:enzymology; D011485:Protein Binding; D014334:Tropolone / Q000031:analogs & derivatives / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008670:Metals; D014764:Viral Proteins; C497842:beta-thujaplicinol; D008345:Manganese; D014334:Tropolone; D004706:Endodeoxyribonucleases; C030894:terminase; D008274:Magnesium; D008239:Lysine