STRUCTURAL-ANALYSIS OF ANTIBODY SPECIFICITY - DETAILED COMPARISON OF 5 FAB'-STEROID COMPLEXES

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-hemisuccinat e, progesterone-11 alpha-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 Ang strom. These different terone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional stru ctures. Comparison of the unliganded DB3 Fab' and these five steroid-F ab' 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 th e binding site is occluded by TrpH100. Small but significant conformat ional changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various crossreactive l igands are accommodated in the binding site in two distinct orientatio ns. We term these binding modes syn and anti, as they are defined by t he orientation of the steroid P face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainl y by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the ster oid in the pocket. The AsnH35 hydrogen bond and the interaction with T rpH50 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 differe nt ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specifi city, such as linker position and the paucity of functional groups on the immunogenic hapten.