Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt-Dependent Thermodynamics

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (> 34 bp) gaps between bound proteins, reversibly bending it by 140o and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around—but not wrapped on—the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log–log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; the most probable values of Ski on 34-bp DNA or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed.

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► Three nonspecific DNA binding modes (6 bp, 10 bp, and 34 bp) of E. coli HUαβ have been identified and characterized by ITC as a function of salt concentration and DNA length.
► Log–log salt concentration derivatives of the binding constants (Ski) for the three modes are found to be the same within experimental uncertainty.
► We conclude that the binding interfaces of all three modes involve the saddle/arm regions of HU and that the 34-bp mode involves bending—but not wrapping—DNA around the body of HU, unlike the wrapped 34-bp complex formed by the structural homolog integration host factor.
► Structural models for the three DNA binding modes are proposed; computational (nonlinear Poisson–Boltzmann) predictions of Ski for these models are the same for all three modes, consistent with experiments.