title: Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.
abstract: The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1) encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+)-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC)/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+)-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Tsung-Fu
authors.initials: TF
authors.lastname: Yu
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Manuel
authors.initials: M
authors.lastname: Maestre-Reyna
authors.affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan; Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Chia-Yun
authors.initials: CY
authors.lastname: Ko
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Tsai-Yun
authors.initials: TY
authors.lastname: Lin
authors.affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan; Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan.
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D056831:Coordination Complexes / Q000737:chemistry; D018360:Crystallography, X-Ray; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004720:Endonucleases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D056831:Coordination Complexes; D004277:DNA, Single-Stranded; D010710:Phosphates; D013431:Sulfates; C574857:ENDO2 protein, Arabidopsis; D004720:Endonucleases; D015032:Zinc