Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana

PLoS One. 2014 Aug 26;9(8):e105821. doi: 10.1371/journal.pone.0105821. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology*
  • Arabidopsis Proteins / chemistry*
  • Catalytic Domain
  • Coordination Complexes / chemistry
  • Crystallography, X-Ray
  • DNA, Single-Stranded / chemistry*
  • Endonucleases / chemistry*
  • Models, Molecular
  • Molecular Sequence Data
  • Phosphates / chemistry
  • Sulfates / chemistry
  • Zinc / chemistry

Substances

  • Arabidopsis Proteins
  • Coordination Complexes
  • DNA, Single-Stranded
  • Phosphates
  • Sulfates
  • ENDO2 protein, Arabidopsis
  • Endonucleases
  • Zinc

Associated data

  • PDB/4CWM
  • PDB/4CXO
  • PDB/4CXP
  • PDB/4CXV

Grants and funding

This work was supported by Academia Sinica, and Ministry of Science and Technology grants: NSC-101-2313-B-214-002-MY3 (to J.-F. Shaw), (NSC-103-3113-P-008-001, and NSC-101-2319-B-001-003 (to A. H.-J. Wang). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.