Thermodynamics of HMGB1 interaction with duplex DNA

Biochemistry. 2001 Aug 28;40(34):10254-61. doi: 10.1021/bi0100900.

Abstract

The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartic Acid
  • Base Pairing
  • Base Sequence
  • Binding Sites
  • Calorimetry
  • Cloning, Molecular
  • DNA / chemistry*
  • DNA / metabolism*
  • Glutamic Acid
  • High Mobility Group Proteins / chemistry*
  • High Mobility Group Proteins / metabolism*
  • Kinetics
  • Models, Molecular
  • Nucleic Acid Conformation
  • Poly dA-dT / chemistry
  • Poly dA-dT / metabolism
  • Polydeoxyribonucleotides / chemistry*
  • Polydeoxyribonucleotides / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Repetitive Sequences, Amino Acid
  • Thermodynamics

Substances

  • High Mobility Group Proteins
  • Polydeoxyribonucleotides
  • Recombinant Proteins
  • Poly dA-dT
  • poly(dC-dG)
  • Aspartic Acid
  • Glutamic Acid
  • DNA