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
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
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DOI：
10.1006/jmbi.2001.4768
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发表时间：
2001-07-06
影响因子：
5.6
通讯作者：
Record, MT
中科院分区：
文献类型：
--
作者：
Holbrook, JA;Tsodikov, OV;Record, MT
结构蛋白整合宿主因子（IHF）的位点特异性DNA结合参与大肠杆菌中功能性多蛋白-DNA组装体的形成，而IHF和其他组蛋白样蛋白的非特异性结合则用于构建类核。在这里，我们报告了一个等温滴定量热法研究结合IHF的热力学的34 bp的片段完全组成的特定H '网站从λ-噬菌体DNA。在低至中等[K+]（60-100 mM）下，由于特异性比低（类似于10（2））和非特异性位点大小非常小，观察到特异性和非特异性结合之间存在强烈竞争。在此[K+]范围内，特异性和非特异性结合都是由焓驱动的，具有大的负焓、熵和热容变化以及对[K+]不敏感的结合常数。高于100 mM K+时，仅观察到特异性结合，并且结合常数以及焓、熵和热容变化的幅度都随着[K+]的增加而强烈降低。热力学为以前未认识设计原理提供了令人信服的证据，通过该原理，与核酸形成广泛结合界面的蛋白质控制结合常数、结合位点大小以及温度和离子浓度对稳定性和特异性的影响。我们提出，位于复合物中DNA磷酸氧原子6埃范围内的23个IHF阳离子侧链中，多达22个在不存在DNA的情况下通过与分子内盐桥（脱水离子对）中的阴离子羧酸盐基团配对而被掩盖。这些盐桥的稳定性随着温度的升高和[K+]的降低而增加。为了解释IHF-DNA相互作用的不寻常的热力学，我们提出，在低[K+]的特异性和非特异性结合需要破坏盐桥（多达18个特异性结合），因此许多未掩蔽的带电基团水合和阳离子基团与DNA相互作用。从结构或热力学与IHF的相似性，我们提出，大规模的耦合破坏的蛋白质盐桥DNA结合是显着的其他大界面的DNA包装蛋白，包括核小体，乳糖阻遏核心四聚体，RNA聚合酶核心蛋白，HU和SSB。(C)北京：科学出版社.
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 titration 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 between 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+] range, 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 observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K+].When interpreted in the context of the structure of the specific complex, the 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 effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic sidechains that are located within 6 Angstrom of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate,groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K+]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K+] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB. (C) 2001 Academic Press.