Thermodynamics of HMGB1 interaction with duplex DNA

The high mobility group protein HMGB1 is a small, highly abundant protein t hat 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 a nd a continuous stretch of 30 glutamate or aspartate residues. Isothermal t itration calorimetry was used to characterize the binding of HMGB 1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To eluc idate the contribution of the different structural motifs to DNA binding, c alorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), an d the full-length HMGB 1 protein. Thermodynamically, binding of HMGB 1 and all truncated constructs to duplex DNA was characterized by a positive enth alpy change at 15 degreesC. From the slopes of the temperature dependence o f the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.1 05 +/- 0.05 kcal mol(-1) K-1 were determined for box A and full-length HMGB 1, respectively. Significant differences in the binding characteristics wer e observed using full-length HMGB1, suggesting an important role for the ac id tail in modulating DNA binding. Moreover, full-length HMGB 1 binds diffe rently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperativ e, had a larger apparent binding site size, and proceeded with a much large r unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).