Arevalo J H, Hassig C A, Stura E A, Sims M J, Taussig M J, Wilson I A
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
J Mol Biol. 1994 Sep 2;241(5):663-90. doi: 10.1006/jmbi.1994.1543.
Structures of the Fab' fragment of the anti-progesterone antibody DB3 in complex with five cross-reactive steroids (aetiocholanolone, 5 beta-androstane-3,17-dione, 5 alpha-pregnane-20-one-3 beta-ol-hemisuccinate, progesterone-11 alpha-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 A. These different steroids compete with progesterone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional structures. Comparison of the unliganded DB3 Fab' and these five steroid-Fab' complexes reveals that all the steroid ligands bind to an "open" conformation of the Fab' as defined by the orientation of the indole side-chain of TrpH100, whereas in the unliganded or "closed" form the binding site is occluded by TrpH100. Small but significant conformational changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various cross-reactive ligands are accommodated in the binding site in two distinct orientations. We term these binding modes syn and anti, as they are defined by the orientation of the steroid beta face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainly by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 alpha position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specificity, such as linker position and the paucity of functional groups on the immunogenic hapten.
已通过X射线晶体学确定了抗孕酮抗体DB3的Fab'片段与五种交叉反应性甾体（本胆烷醇酮、5β-雄甾烷-3,17-二酮、5α-孕烷-20-酮-3β-醇半琥珀酸酯、孕酮-11α-醇半琥珀酸酯和孕酮）形成复合物的结构，最高分辨率为2.7埃。尽管这些不同甾体的三维结构存在显著差异，但它们与孕酮结合的亲和力在纳摩尔范围内相互竞争。未结合配体的DB3 Fab'与这五种甾体-Fab'复合物的比较表明，所有甾体配体均结合至由TrpH100吲哚侧链方向所定义的Fab'“开放”构象，而在未结合配体或“封闭”形式中，结合位点被TrpH100封闭。抗体发生微小但显著的构象变化，以最大程度地实现与每种配体的物理和化学互补性。各种交叉反应性配体以两种不同方向容纳于结合位点。我们将这些结合模式称为顺式和反式，因为它们由甾体β面相对于TrpH50的方向所定义。在所有情况下，甾体D环均插入主要由TrpH50、TyrH97、TrpH100和PheH100b形成的疏水腔中；与AsnH35至C17或C20位置酮基的氢键相互作用使甾体在口袋中定向。AsnH35氢键以及与TrpH50的相互作用解释了重链对在11α位置衍生的孕酮样甾体免疫反应的受限情况。抗体与不同甾体的交叉反应性可通过A环的替代结合口袋来解释，这在结合位点产生了不同的配体方向。这项研究表明了哪些因素最有可能导致所观察到的抗体特异性，例如连接子位置以及免疫原性半抗原上官能团的缺乏。
