Herron J N, Terry A H, Johnston S, He X M, Guddat L W, Voss E W, Edmundson A B
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City 84112.
Biophys J. 1994 Dec;67(6):2167-83. doi: 10.1016/S0006-3495(94)80738-6.
Three-dimensional structures were determined for three crystal forms of the antigen binding fragment (Fab) of anti-fluorescein antibody 4-4-20 in complex with fluorescein. These included 1) a triclinic (P1) form crystallized in 47% (v/v) 2-methyl-2,4-pentanediol (MPD); 2) a triclinic (P1) form crystallized in 16% (w/v) poly(ethylene glycol), molecular weight 3350 (PEG); and 3) a monoclinic (P21) form crystallized in 16% PEG. Solvent molecules were added to the three models and the structures were refined to their diffraction limits (1.75-A, 1.78-A, and 2.49-A resolution for the MPD, triclinic PEG, and monoclinic PEG forms, respectively). Comparisons of these structures were interesting because 4-4-20 exhibited a lower antigen-binding affinity in 47% MPD (Ka = 1.3 x 10(8) M-1) than in either 16% PEG (Ka = 2.9 x 10(9) M-1) or phosphate-buffered saline (Ka = 1.8 x 10(10) M-1). Even though the solution behavior of the antibody was significantly different in MPD and PEG, the crystal structures were remarkably similar. In all three structures, the fluorescein-combining site was an aromatic slot formed by tyrosines L32, H96, and H97 and tryptophans L96 and H33. In addition, several active site constituents formed an electrostatic network with the ligand. These included a salt link between arginine L34 and one of fluorescein's enolate oxygen atoms, a hydrogen bond between histidine L27d and the second enolic group, a hydrogen bond between tyrosine L32 and the phenylcarboxylate group, and two medium range (approximately 5 A) electrostatic interactions with lysine L50 and arginine H52. The only major difference between the triclinic MPD and PEG structures was the degree of hydration of the antigen-combining site. Three water molecules participated in the above electrostatic network in the MPD structure, while eight were involved in the PEG structure. Based on this observation, we believe that 4-4-20 exhibits a lower affinity in MPD due to the depletion of the hydration shell of the antigen-combining site.
测定了抗荧光素抗体4-4-20的抗原结合片段（Fab）与荧光素形成的三种晶体形式的三维结构。其中包括：1）在47%（v/v）2-甲基-2,4-戊二醇（MPD）中结晶的三斜晶系（P1）形式；2）在16%（w/v）分子量为3350的聚乙二醇（PEG）中结晶的三斜晶系（P1）形式；3）在16% PEG中结晶的单斜晶系（P21）形式。向这三种模型中添加了溶剂分子，并将结构精修至其衍射极限（MPD、三斜晶系PEG和单斜晶系PEG形式的分辨率分别为1.75 Å、1.78 Å和2.49 Å）。这些结构的比较很有趣，因为4-4-20在47% MPD中的抗原结合亲和力（Ka = 1.3×10⁸ M⁻¹）低于在16% PEG（Ka = 2.9×10⁹ M⁻¹）或磷酸盐缓冲盐溶液（Ka = 1.8×10¹⁰ M⁻¹）中的亲和力。尽管抗体在MPD和PEG中的溶液行为有显著差异，但晶体结构却非常相似。在所有三种结构中，荧光素结合位点是由酪氨酸L32、H96和H97以及色氨酸L96和H33形成的芳香族缝隙。此外，几个活性位点成分与配体形成了一个静电网络。其中包括精氨酸L34与荧光素的一个烯醇氧原子之间的盐键、组氨酸L27d与第二个烯醇基团之间的氢键、酪氨酸L32与苯甲酸盐基团之间的氢键，以及与赖氨酸L50和精氨酸H52的两个中等距离（约5 Å）的静电相互作用。三斜晶系MPD和PEG结构之间唯一的主要差异是抗原结合位点的水合程度。在MPD结构中，三个水分子参与了上述静电网络，而在PEG结构中有八个水分子参与。基于这一观察结果，我们认为4-4-20在MPD中亲和力较低是由于抗原结合位点水合壳层的消耗。
