The Distribution of Fatty Acids Reveals the Functional Structure of Human Serum Albumin
The Distribution of Fatty Acids Reveals the Functional Structure of Human Serum Albumin
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DOI：
10.1002/anie.201003495
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发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Hinderberger, Dariush
中科院分区：
文献类型：
--
作者：
Junk, Matthias J. N.;Spiess, Hans Wolfgang;Hinderberger, Dariush
人血清白蛋白(HSA)是人体血浆中含量最丰富的蛋白质，是多种内源性化合物和药物分子的转运体。[1，2]其结合和转运多种脂肪酸(FA)的能力已被广泛研究。[3，4]HSA的研究受到蛋白质复杂性的严重阻碍，并从晶体高分辨结构中获益良多。大约20年前，他和卡特报道了HSA的第一个晶体结构。[5]到目前为止，蛋白质数据库中已经存储了大量的HSA晶体结构。对于了解蛋白质的结合特性，更重要的是了解HSA和运输分子的络合物的结构。由于Curry等人的开创性工作，各种HSA/脂肪酸复合体的晶体结构已经变得可用。[6-8]他们发现脂肪酸在蛋白质晶体中高度不对称分布，尽管HSA本身显示出对称的一级和二级结构。发现了长链脂肪酸的多达七个不同的结合部位，其中大多数由离子锚定单元和长长的疏水口袋组成。[8，9]两到三个高亲和力结合部位[3，10]的位置是通过X射线结构与取代13C标记脂肪酸的药物竞争结合的核磁共振研究确定的。[11，12]第2、4和5位与脂肪酸具有高亲和力，而1、3、6和7位对脂肪酸的亲和力略低(见图1a)。更广泛地说，蛋白质晶体结构在多大程度上反映了蛋白质在溶液中的动态和功能结构，这是一个长期存在的争论。X射线结晶学数据与基于溶液状态的技术(如核磁共振和其他类型的光谱以及中子散射)或分子动力学模拟的结果之间的明显差异往往会加剧这场辩论。此外，人们越来越意识到蛋白质在溶液中的动态与生物功能有关。最近的核磁共振研究表明，许多蛋白质表现出明显的动态构象灵活性，这对功能有影响。[13-15]在这方面，我们注意到HSA的表面暴露部分显示出高度的灵活性，这构成了该蛋白质在结合各种分子时的多功能性的关键。早在20世纪50年代，Karush就提出了一个解释结合位点的构象适应性的概念。[16，17]此外，还提出了一个考虑脂肪酸烷链柔性引起的构象熵的模型。[18]我们的研究旨在从脂肪酸的角度直接揭示HSA与脂肪酸结合的功能结构。这是通过电子顺磁共振(EPR)光谱实现的，研究含有稳定的氮氧自由基的自旋标记脂肪酸并产生EPR信号。因此，检测到FA结合位点的分布而没有来自复合蛋白本身的任何贡献(图1)。结合部位的结构信息是通过测定冷冻过程中脂肪酸之间的距离分布来获得的
Human serum albumin (HSA), the most abundant protein in human blood plasma, serves as a transporting agent for various endogenous compounds and drug molecules.[1, 2] Its capability to bind and transport multiple fatty acids (FA), in particular, has been studied extensively in the past.[3, 4] Research on HSA was severely hampered by the complexity of the protein and has benefitted tremendously from crystallographic high-resolution structures. Nearly 20 years ago, He and Carter reported the first crystal structure of HSA.[5] To date, a multitude of HSA crystal structures have been deposited in the Protein Data Bank. Even more important for understanding the binding properties of the protein are the structures of complexes of HSA and transported molecules. Thanks to the pioneering work of Curry et al., crystal structures of various HSA/fatty acid complexes have become accessible.[6–8] They found that fatty acids are distributed highly asymmetrically in the protein crystal although HSA itself exhibits a symmetric primary and secondary structure. Up to seven distinct binding sites were found for long-chain fatty acids, most of which comprised ionic anchoring units and long, hydrophobic pockets.[8, 9] The location of two to three high-affinity binding sites [3, 10] was assigned by correlation of the X-ray structure with NMR studies on competitive binding of drugs that replaced 13C-labeled fatty acids.[11, 12] Sites 2, 4, and 5 bind fatty acids with a high affinity, while sites 1, 3, 6, and 7 exhibit a somewhat lower affinity to fatty acids (see Figure 1a). More generally, there is a long-standing debate as to what extent protein crystal structures reflect the dynamic and functional structures of proteins in solution. This debate is often fueled by apparent discrepancies between X-ray crystallographic data and results from solution-state-based techniques (eg NMR and other types of spectroscopy as well as neutron scattering) or from molecular dynamics simulations. Moreover, there is an increasing awareness that protein dynamics in solution is connected to biological function. Recent NMR studies revealed that many proteins exhibit pronounced dynamic conformational flexibilities with implications for the function.[13–15] In this context we note that the surface-exposed parts of HSA show a high degree of flexibility which constitutes a key to the versatility of this protein in binding various molecules. Already in the 1950s, Karush developed a concept that accounted for this conformational adaptability of the binding sites.[16, 17] Further, a model has been proposed that takes into account the conformational entropy arising from the flexibility of the fatty acid alkyl chains.[18] Our study aims at revealing the functional structure of HSA with respect to its binding of fatty acids directly from the fatty acids point of view. This is achieved by electron paramagnetic resonance (EPR) spectroscopy, studying spinlabeled fatty acids that have stable nitroxide radicals incorporated and give rise to an EPR signal. Thus, the distribution of the FA binding sites is detected without any contribution from the complex protein itself (Figure 1). Structural information on the binding sites is obtained by determining the distance distributions between the fatty acids in frozen