THE RATIONAL CONSTRUCTION OF AN ANTIBODY AGAINST CYSTATIN - LESSONS FROM THE CRYSTAL-STRUCTURE OF AN ARTIFICIAL F-AB FRAGMENT

Ln a protein design study the artificial antibody M41 was modelled wit h its binding site complementary to the protease inhibitor cystatin, w hich was chosen as a structurally well-characterized ''antigen''. The modelling of M41 took advantage of the crystal structure of the anti-l ysozyme antibody HyHEL-10 as a structural template. Its combining site was reshaped by replacing 19 amino acid side-chains in the hypervaria ble loops. Ln addition, ten amino acid residues were substituted in th e framework regions. The crystal structure of the corresponding antibo dy model M41, which was produced as an F-ab fragment in Escherichia co li, was determined at a resolution of 1.95 Angstrom. The crystals exhi bited symmetry of the space group P2(1)2(1)2(1) (a = 96.5 Angstrom; b = 103.5 Angstrom; c = 113.6 Angstrom) with two F-ab fragments in the a symmetric unit, which were independently refined (final R-factor 21.7% ). The resulting coordinates were used for a detailed comparison with the modelled protein structure. It was found that the mutual arrangeme nt of the six complementarity-determining regions as well as most of t heir backbone conformation had been correctly predicted. One major dif ference that was detected for the conformation of a five residue inser tion in complementarity-determining region L1 could be explained by an erroneously defined segment in the structure of the antibody 4-4-20, which had been used as a template for this loop. In the light of more recent crystallographic data it appears that this segment adopts a new canonical structure. Apart from this region, most of the side-chains in the antigen-binding site had been properly placed in the M41 model. There was however one important exception concerning Try H98, whose s ide-chain conformation had been kept as it appeared in HyHEL-10. The d iffering orientation of this residue in the model compared with the cr ystal structure of the artificial F-ab fragment M41 explains why an an tigen affinity could not be detected so far. The detailed analysis of this and other, more subtle deviations suggests how to make this F-ab fragment function by introducing a few additional amino acid changes i nto M41. (C) 1997 Academic Press Limited.