Specific and non-specific interactions of integration host factor with DNA: Thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal t itration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H ' site from lambda -phage DNA. At low to moderate [K+] (60-100 mM), strong competition is observed betwee n specific and non-specific binding as a result of a low specificity ratio (similar to 10(2)) and a very small non-specific site size. In this [K+] ra nge, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K+]. Above 100 mM K+ only specific binding is obs erved, and both the binding constant and the magnitudes of enthalpy, entrop y and heat capacity changes all decrease strongly with increasing [K+]. When interpreted in the context of the structure of the specific complex, t he thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effect s of temperature and ion concentrations on stability and specificity. We pr opose that up to 22 of the 23 IHF cationic sidechains that are located with in 6 Angstrom of DNA phosphate oxygen atoms in the complex, are masked in t he absence of DNA by pairing with anionic carboxylate,groups in intramolecu lar salt-bridges (dehydrated ion-pairs). These salt-bridges increase in sta bility with increasing temperature and decreasing [K+]. To explain the unus ual thermodynamics of IHF-DNA interactions, we propose that both specific a nd non-specific binding at low [K+] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged gro ups hydrate and the cationic groups interact with DNA. From structural or t hermodynamic parallels with IHF, we propose that large-scale coupling of di sruption of protein salt-bridges to DNA binding is significant for other la rge-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB. (C) 2001 Academic Press.