Determination of the binding specificity of an integral membrane protein by saturation transfer difference NMR:: RGD peptide ligands binding to integrin αIIbβ3
Determination of the binding specificity of an integral membrane protein by saturation transfer difference NMR:: RGD peptide ligands binding to integrin αIIbβ3
复制标题
DOI：
10.1021/jm0109154
复制
发表时间：
2001-09-13
影响因子：
7.3
通讯作者：
Meyer, B
中科院分区：
文献类型：
--
作者：
Meinecke, R;Meyer, B
饱和转移差（STD）NMR是一种快速和通用的方法，用于在受体存在下筛选化合物混合物的结合亲和力，并表征配体的结合表位。在这里，我们表明，如果受体嵌入到脂质体的脂质双层中，则可以通过STD NMR来研究配体与膜蛋白的相互作用。整合素α（IIb）β（3），也称为GPIIb-IIIa，是血小板表面糖蛋白，其在血小板聚集中起关键作用，并与呈递肽识别基序RGD的蛋白质和肽相互作用。将纯化的人整合素α（IIb）β（3）掺入脂质体中，并通过STD NMR技术分析RGD肽的结合。Cyclo（RGDfV）在含有整联蛋白的脂质体存在下产生STD NMR效应。STD效应的大小作为配体浓度的函数给出了30-60 μ M的解离常数值。将弱结合的RGD添加到环（RGDfV）的溶液中导致仅较强配体环（RGDfV）的STD效应。这证明与文献一致，肽RGD是比肽环（RGDfV）弱得多的整联蛋白配体，肽环（RGDfV）在很大程度上取代了来自结合位点的RGD肽。通过STD NMR表征配体环状（RGDfV）的结合表位，其包含D-Phe、瓦尔甲基、Arg α、β和γ质子、Asp的一个H β和Gly的一个H α部分。
Saturation transfer difference (STD) NMR is a fast and versatile method to screen compound mixtures in the presence of a receptor for binding affinity and to characterize the ligand's binding epitope. Here we demonstrate that ligand interactions with integral membrane proteins can be investigated by STD NMR if the receptor is embedded into the lipid bilayer of a liposome. The integrin alpha (IIb)beta (3), also termed GPIIb-IIIa, is a platelet surface glycoprotein that plays a pivotal role in platelet aggregation and that interacts with proteins and peptides presenting the peptide recognition motif RGD, Purified human integrin alpha (IIb)beta (3) was incorporated into liposomes, and the binding of RGD peptides was analyzed by STD NMR techniques. Cyclo(RGDfV) gave STD NMR effects in the presence of liposomes containing the integrin. The magnitude of the STD effect as a function of the ligand's concentration gave a value for the dissociation constant of 30-60 muM. Adding the weakly binding RGD to the solution of cyclo(RGDfV) resulted in STD effects of the stronger ligand cyclo(RGDfV) only. This demonstrates in agreement with literature that the peptide RGD is a much weaker ligand to the integrin than the peptide cyclo(RGDfV) that largely replaces the RGD peptides from the binding site. The binding epitope of the ligand cyclo(RGDfV) was characterized by STD NMR to contain sections of the D-Phe, the Val methyl groups, Arg alpha, beta, and gamma protons, one H beta of Asp, and one H alpha of Gly.