Institut de Biologie Structurale, CNRS, CEA, University Grenoble Alpes, UMR 5075, 41 rue Horowitz, 38027, Grenoble cedex 01, France.
J Am Chem Soc. 2013 Jun 26;135(25):9384-90. doi: 10.1021/ja4000867. Epub 2013 Jun 13.
The extensive functional repertoire of heparin and heparan sulfate, which relies on their ability to interact with a large number of proteins, has recently emerged. To understand the forces that drive such interactions the binding of heparin to interferon-γ (IFNγ), used as a model system, was investigated. NMR-based titration experiments demonstrated the involvement of two adjacent cationic domains (D1: KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal sequence of the cytokine. Kinetic analysis showed that these two domains contribute differently to the interaction: D1 is required to form a complex and constitutes the actual binding site, whereas D2, although unable to associate with heparin by itself, increased the association rate of the binding. These data are consistent with the view that D2, through nonspecific electrostatic forces, places the two molecules in favorable orientations for productive binding within the encounter complex. This mechanism was supported by electrostatic potential analysis and thermodynamic investigations. They showed that D1 association to heparin is driven by both favorable enthalpic and entropic contributions, as expected for a binding sequence, but that D2 gives rise to entropic penalty, which opposes binding in a thermodynamic sense. The binding mechanism described herein, by which the D2 domain kinetically drives the interaction, has important functional consequences and gives a structural framework to better understand how specific are the interactions between proteins and heparin.
肝素和硫酸乙酰肝素具有广泛的功能，这依赖于它们与大量蛋白质相互作用的能力，这种能力最近才被发现。为了了解驱动这些相互作用的力，研究了肝素与干扰素-γ（IFNγ）的结合，IFNγ 被用作模型系统。基于 NMR 的滴定实验表明，两个相邻的阳离子结构域（D1：KTGKRKR 和 D2：RGRR）都参与了细胞因子的羧基末端序列，参与了这种相互作用。动力学分析表明，这两个结构域对相互作用的贡献不同：D1 是形成复合物所必需的，构成了实际的结合位点，而 D2 虽然不能单独与肝素结合，但增加了结合的结合速率。这些数据与 D2 通过非特异性静电力将两个分子置于有利于在遭遇复合物中进行有效结合的有利取向的观点一致。静电势能分析和热力学研究支持了这一机制。它们表明，D1 与肝素的结合是由有利的焓和熵贡献驱动的，这与结合序列相符，但 D2 导致了熵罚，从热力学意义上反对结合。本文描述的结合机制，即 D2 结构域通过动力学驱动相互作用，具有重要的功能意义，并为更好地理解蛋白质与肝素之间的相互作用的特异性提供了结构框架。
