Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy
Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy
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DOI：
10.1073/pnas.0506440102
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发表时间：
2005-12-13
影响因子：
11.1
通讯作者：
Hamilton, JA
中科院分区：
文献类型：
--
作者：
Simard, JR;Zunszain, PA;Hamilton, JA
人血清白蛋白（HSA）是一种多用途的内源性化合物和药物转运蛋白。为了评估蛋白质配体之间的生理相关相互作用，有必要确定不同配体的结合位点的位置和相对亲和力。我们提出了一项位点特异性的研究，研究了HSA上脂肪酸（FA）结合位点的相对亲和力，FA是蛋白质的主要生理配体。最初使用[C-13]羧基标记的FA滴定HSA，以确定与蛋白质上高亲和力结合口袋相关的三种核磁共振化学位移。为了将这些峰与从FA-HSA复合物的晶体结构中鉴定出的FA结合位点联系起来，HSA突变体被替换为参与结合FA羧基配位的氨基酸。将[C-13]棕榈酸酯滴定到HSA突变体的溶液中，无论是在HSA III结构域内的FA位点4 （R410A/Y411A）还是位点5 (K525A)，都显示出在野生型蛋白光谱中存在的特定NMR峰的丢失。因为这些峰是在用棕榈酸滴定HSA时最先观察到的三个峰之一，所以位点4和5代表了HSA上三个高亲和长链fa结合位点中的两个。通过将[C-13]棕榈酸酯滴定到HSA重组结构域III中证实了这些定位，重组结构域III只包含4位和5位。这些结果为直接探测FA结合位点的相对亲和力建立了一个协议，可以扩展到研究FA和其他配体之间对特定结合位点的竞争。
Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [C-13]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA-HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [C-13]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [C-13]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites.