Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis
Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis
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DOI：
10.1016/j.jmb.2006.06.028
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发表时间：
2006-08-11
影响因子：
5.6
通讯作者：
Curry, Stephen
中科院分区：
文献类型：
--
作者：
Simard, Jeffrey R.;Zunszain, Patricia A.;Curry, Stephen
人血清白蛋白(HSA)是一种丰富的血浆蛋白，可运输多种药物和内源性化合物。HSA的复杂结合能力使其成为一个具有挑战性的详细研究系统，但为了加深我们对HSA配体之间相互作用的理解，必须确定不同配体结合部位的位置和相对亲和力。白蛋白与其主要的生理配体--非酯化脂肪酸(FA)具有多个结合部位。以前，用C-13标记的FA滴定BSA显示了多个化学位移，并允许识别与高亲和力FA结合相关的三个化学位移的子集。最近的人血清白蛋白的结晶学研究已经绘制出至少七个长链FA的FA结合位点，并描绘了与药物和其他内源化合物的结合位点的重叠。我们的目标是将核磁共振和FA的结构数据关联起来，以提供更完整的HSA结合能力的描述。我们最近的诱变研究使我们能够在HSA的结构域III中鉴定出两个高亲和力结合位点。在这里，我们用核磁共振研究了C-13-羧基标记棕榈酸酯在竞争配体存在和不存在的情况下与人血清白蛋白的结合，以完成核磁共振化学位移与特定结构结合位点的关联。我们仔细选择了具有特定结合位置的配体，并使用它们单独或组合与[C-13]棕榈酸酯竞争与HSA的结合。我们发现，FA的2，4和5位与FA具有高亲和力，而1，3，6和7位对FA的亲和力较低，从而首次完整地确定了所有7个长链FA在HSA上的相对亲和力。我们的结果也为FA与其他配体之间的相互作用提供了直接的见解。(C)2006爱思唯尔有限公司。保留所有权利。
Human serum albumin (HSA) is an abundant plasma protein that transports a wide variety of drugs and endogenous compounds. The complex binding capacity of HSA has made it a challenging system to study in detail but in order to develop our understanding of the interactions between ligands for HSA, the locations and relative affinities of different ligand binding sites must be determined. Albumin possesses multiple binding sites for its primary physiological ligand, non-esterified fatty acids (FA). Previously, titration of BSA with C-13-labeled FA revealed multiple chemical shifts and allowed identification of a subset of three chemical shifts that were associated with high affinity FA binding. Recent crystallographic studies of HSA have mapped at least seven FA binding sites for long-chain FA and delineated the overlap with binding sites for drugs and other endogenous compounds. We aim to correlate NMR and structural data for FA to provide a more complete description of the binding capacity of HSA. Our recent mutagenesis studies allowed us to identify two high affinity binding sites in domain III of HSA. Here, we use NMR to study the binding of C-13-carboxyl labeled palmitate to HSA in the presence and absence of competitor ligands to complete the correlation of NMR chemical shifts with specific structural binding sites. We carefully selected ligands with specific binding sites identified by crystallogaphy and used them, either singly or in combination, to compete with [C-13]palmitate for binding to HSA. We show that FA sites 2,4 and 5 bind FA with high affinity, while sites 1, 3,6 and 7 exhibit low affinity for FA, thus providing the first complete determination of relative affinities of all seven long-chain FA sites on HSA. Our results also yield direct insights into the interactions between FA and other ligands. (c) 2006 Elsevier Ltd. All rights reserved.