Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase

Robert L. Crowther, David P. Remeta, Conceição A S A Minetti, Debanu Das, Sherwin P. Montano, Millie Georgiadis

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have previously reported that processive DNA synthesis of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is severely compromised by substitution of an Ala for the fingers domain residue Arg 116. In order to further investigate the role of Arg 116 in interactions of MMLV RT with nucleic acids, we have determined the crystal structure of the R116A N-terminal fragment and characterized the binding of two self-complementary DNA duplexes [d(CATGCATG)2 and d(CGCGCGCG)2] to both the wild-type and R116A fragments by isothermal titration calorimetry. The resultant thermodynamic profiles extrapolated to 25°C reveal that binding of the wild-type N-terminal fragment to both DNA duplexes is enthalpy-driven and characterized by an unfavorable entropy. Although the temperature dependence of the respective protein-DNA binding enthalpies is markedly different reflecting distinct heat capacity changes, the binding free energies are nearly identical and relatively invariant to temperature (ΔG ∼ -6.0 kcal · mol-1). In contrast to the wild-type fragment, the R116A fragment exhibits no measurable affinity for either DNA duplex, yet its crystal structure reveals no significant changes when compared to the wild-type structures. We suggest that hydrogen-bonding interactions involving the fingers domain residue Arg 116 are critical for DNA binding as well as processive DNA synthesis by MMLV RT.

Original languageEnglish
Pages (from-to)15-26
Number of pages12
JournalProteins: Structure, Function and Genetics
Volume57
Issue number1
DOIs
StatePublished - Oct 1 2004

Fingerprint

Moloney murine leukemia virus
RNA-Directed DNA Polymerase
Viruses
Nucleic Acids
Fingers
DNA
Enthalpy
Genes
Crystal structure
Genome
Calorimetry
Temperature
DNA-Binding Proteins
Entropy
Retroviridae
Hydrogen Bonding
Titration
Thermodynamics
Free energy
Specific heat

Keywords

  • Energetics
  • MMLV reverse transcriptase
  • Mutagenesis
  • Processivity
  • Protein-DNA interactions
  • Structure

ASJC Scopus subject areas

  • Genetics
  • Structural Biology
  • Biochemistry

Cite this

Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase. / Crowther, Robert L.; Remeta, David P.; Minetti, Conceição A S A; Das, Debanu; Montano, Sherwin P.; Georgiadis, Millie.

In: Proteins: Structure, Function and Genetics, Vol. 57, No. 1, 01.10.2004, p. 15-26.

Research output: Contribution to journalArticle

Crowther, Robert L. ; Remeta, David P. ; Minetti, Conceição A S A ; Das, Debanu ; Montano, Sherwin P. ; Georgiadis, Millie. / Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase. In: Proteins: Structure, Function and Genetics. 2004 ; Vol. 57, No. 1. pp. 15-26.
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