Thermodynamics of HMGB1 interaction with duplex DNA.
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).
Müller
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DIBIT, San Raffaele Scientific Institute, via Olgettina 58, 2032 Milano, Italy.
Bianchi
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Knapp
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D001224:Aspartic Acid; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011067:Poly dA-dT / Q000737:chemistry / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013816:Thermodynamics
D006609:High Mobility Group Proteins; D011089:Polydeoxyribonucleotides; D011994:Recombinant Proteins; D011067:Poly dA-dT; C019530:poly(dC-dG); D001224:Aspartic Acid; D018698:Glutamic Acid; D004247:DNA