Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.

中文翻译：

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抗体识别聚乙二醇的结构基础

聚乙二醇（PEG）广泛用于工业和医学。已经开发出抗PEG抗体用于表征PEG化药物和其他应用。然而，尚未阐明特异性PEG结合的潜在机制。将两种同源抗PEG抗体3.3和2B5的Fab各自与PEG复合结晶，并通过X射线衍射确定其结构。分析了这两个晶体中的PEG-Fab相互作用并将其与不相关的抗血凝素32D6-Fab的含PEG晶体中的相互作用进行了比较。通过使用分析超速离心机（AUC）检查PEG结合化学计量。可以看到一个常见的与3.3和2B5的PEG结合模式，其中一个S型核心PEG片段与两个与dyad相关的Fab分子结合。附近的卫星结合位点可能容纳更长的PEG分子的一部分。核心PEG片段主要与重链残基D31，W33，L102，Y103和Y104相互作用，与芳族侧链发生广泛接触。在S形PEG的每个半圆的中心，一个水分子与醚氧原子交替形成氢键，其结构类似于与冠醚键合的赖氨酸。每个卫星片段都夹在两个精氨酸残基之间，分别来自重链的R52和来自轻链的R29，并且还与数个芳族侧链相互作用。相反，在32D6-Fab晶体中非特异性结合的PEG片段位于肘部区域或晶格接触处。AUC数据表明3.3-Fab在无PEG溶液中作为单体存在，但在PEG-550-MME存在时形成二聚体，其大小约为S形核心PEG片段的大小。3.3和2B5中的不同氨基酸不参与PEG结合，但参与二聚体形成。特别地，2B5-Fab的轻链残基K53在二聚体中与另一个Fab显着接触，而3.3-Fab的相应N53没有。二聚体中两个Fab分子之间的蛋白质-蛋白质相互作用的这种差异可能解释了2B5在PEG结合中的温度依赖性，以及其被冠醚的抑制作用。