Solution NMR studies of Chlorella virus DNA ligase-adenylate

J Mol Biol. 2010 Jan 15;395(2):291-308. doi: 10.1016/j.jmb.2009.11.007. Epub 2009 Nov 11.


DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3'-OH/5'-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone (15)N spin relaxation and (15)N,(1)H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5'-phosphate.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Monophosphate / chemistry
  • Adenosine Monophosphate / metabolism
  • Catalytic Domain
  • DNA / genetics
  • DNA / metabolism
  • DNA Ligases / chemistry*
  • DNA Ligases / genetics
  • DNA Ligases / metabolism
  • Kinetics
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Paramecium / virology
  • Phosphates / chemistry
  • Phosphates / metabolism
  • Protein Conformation
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity
  • Thermodynamics
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism


  • Phosphates
  • Recombinant Fusion Proteins
  • Viral Proteins
  • Adenosine Monophosphate
  • DNA
  • Chlorella virus DNA ligase
  • DNA Ligases