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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.
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