ANTIBODY FRAGMENT FV4155 BOUND TO 2 CLOSELY-RELATED STEROID-HORMONES - THE STRUCTURAL BASIS OF FINE SPECIFICITY

Background: The concentration of steroid glucuronides in serial sample s of early morning urine (EMU) can be used to predict the fertile peri od in the female menstrual cycle. The monoclonal antibody 4155 has bee n used as a convenient means of measuring the concentration of steroid glucuronides in EMU, as it specifically recognises the steroid hormon e estrone beta-D-glucuronide (E3G), with very high affinity, and the c losely related hormone estriol 3-(beta-D-glucuronide) (EI3G), with red uced affinity, Although 4115 binds these hormones with different affin ities, EI3G differs from E3G only in the addition of a hydroxyl group and reduction of an adjacent carbonyl. To investigate the structural b asis of this fine binding specificity, we have determined the crystal structures of the variable fragment (Fv) of 4155 in complex with each of these hormones.Results: Two crystal forms of the Fv4155-EI3G comple x, at resolutions of 2.1 Angstrom and 2.5 Angstrom, and one form of th e Fv4155-E3G complex, at 2.1. Angstrom resolution were solved and refi ned. The crystal structures show the E3G or EI3G antigen lying in an e xtended cleft, running from the centre of the antibody combining site down one side of the variable domain interface, and formed almost enti rely from residues in the heavy chain. The binding cleft lies primaril y between the heavy chain complementarity determining regions (CDRs), rather than in the interface between the heavy and light chains, In bo th complexes the binding of the glucuronic sugar, and rings A and B of the steroid, is specified by the shape of the narrow cleft. Analysis of the Fv structure reveals that five of the six CDR regions can be as signed to one of the predefined canonical structural classes. Conclusi ons: The difference in the binding affinity of Fv4155 for the two ster oid hormones is accounted for by a subtle combination of a less favour ed hydrogen-bond geometry, and a minor rearrangement of the water mole cule network around the binding site. The rearrangement of water molec ules results from the burial of the additional hydroxyl group of the E I3G in a hydrophobic environment.