Structural and Thermodynamic Determinants of Anti-PGE2 mAb Specificity

We investigated the molecular determinants underlying the highly specific recognition of prostaglandin (PG) E2 and its structural analogues by an anti-PGE2 monoclonal antibody (mAb) and its Fab fragment using high-resolution X-ray crystallography coupled with detailed thermodynamic analyses, including conditions that mimic macromolecular crowding. Crystal structures of the Fab fragment in complex with PGE2, PGE1, and ONO5 were determined at 1.9 [A], 1.7 [A], and 1.7 [A] resolution, respectively, and the antigen-free Fab structure was solved at 2.0 [A]. The Fab accommodates the L-shaped PG scaffold within a deep antigen-binding crevasse formed between the {beta}-sheet frameworks of the VH and VL domains, where ligand recognition is mediated by multiple water-bridged hydrogen bonds and extensive hydrophobic contacts, including putative CH-O and CH-{pi} interactions. Comparison of antigen-free and ligand-bound structures revealed compensatory conformational rearrangements accompanied by reorganization of bound water networks. Upon PG binding, a short 310 helix forms at the tip of CDR-H1, stabilized by a cluster of hydrogen bonds and capping water molecules. This helix formation is associated with rotation of the VL domain and an increased Fab elbow angle. The CDR-H1 loop undergoes a flip-in transition in which Phe29H inserts into a hydrophobic cavity while Glu31H is extruded toward solvent, accompanied by {pi}-{pi} stacking between Tyr27H and Tyr32H. These changes collectively reduce hydrophobic surface area and promote enthalpy-driven ligand binding. The flexible -chain of PGE1, lacking the C5-C6 double bond, adopts altered conformations that disrupt C1-carboxyl interactions with three aromatic residues and lead to closure of a secondary water channel, consistent with subtle but meaningful differences in binding thermodynamics and crystallographic temperature factors relative to PGE2 and ONO5. Isothermal titration calorimetry (ITC) revealed high-affinity, enthalpy-driven PG binding to the Fab, in agreement with differential scanning calorimetry (DSC), which showed increased thermal stability upon complex formation. Under macromolecular crowding conditions induced by high bovine serum albumin (BSA) concentrations, PG binding to the intact mAb--but not to the Fab--exhibited improved discrimination between PGE2 and PGE1, suggesting that reduced entropy-enthalpy compensation under crowding amplifies subtle structural mismatches in ligand binding.