The de novo design of an antibody combining site: Crystallographic analysis of the V_L domain confirms the structural model

In a protein design approach the molecular model of an artificial antibody F_v fragment was generated with predicted complementarity to part of the known crystal structure of chicken egg-white cystatin. The model of the F_v fragment was based on the three-dimensional structure of the anti-lysozyme antibody HyHEL-10, which was modified by substituting amino acid side-chains in the complementarity-determining regions (CDRs) as well as the framework without altering the backbone. In the course of crystallization experiments with the bacterially produced F_v fragment crystals of the V_L domain alone were obtained. These crystals diffracted X-rays to a resolution of 2·17 Å and were shown to belong to the space group P2₁2₁2₁ with unit cell dimensions a = 46·89 Å, b = 58·05 Å, c = 83·22 Å containing two V_L monomers in the asymmetric unit. The crystal structure was solved by molecular replacement and refined to a crystallographic R-factor of 17.5%. The two V_L monomers exhibit an asymmetric mode of association, which is different from other crystallized V_L domains described before and shows the peculiar feature of an isopropanol precipitant molecule buried at the interface. Both V_L structures reveal a high level of similarity to the predicted three-dimensional model. With the exception of two loop segments in the framework region that are involved in crystal packing contacts, the backbone structures of the two V_L monomers in the crystal and the molecular model of the V_L domainare practically identical. Although six amino acid residues had been replaced in the hypervariable regions, the CDR conformations remained conserved and only minor deviations in the orientation of some side-chains and peptide planes were detected. The crystallographic analysis of the V_L domain modelled aspart of a complex between an artificial F_v fragment and the small protein cystatin, deliberatelychosen as antigen target, confirms the concept of distinct structural classes for CDR backbones and supports our strategy for the de novo design of an antibodycombining site.