The 1.85 angstrom resolution crystal structures of tissue factor in complex with humanized Fab D3h44 and of free humanized Fab D3h44: Revisiting the solvation of antigen combining sites

The outstanding importance of the anti-en-antibody recognition process for the survival and defence strategy of higher organisms is in sharp contrast to the limited high resolution structural data available on antibody-antige n pairs with antigenic proteins. The limitation is the most severe for stru ctural data not restricted to the antigen-antibody complex but extending to the uncomplexed antigen and antibody. We report the crystal structure of t he complex between tissue factor (TF) and the humanized Fab fragment D3h44 at a resolution of 1.85 Angstrom together with the, structure of uncomplexe d D3h44 at the same resolution. In conjunction, with the previously reporte d 1.7 Angstrom crystal structure of uncomplexed TF, a unique opportunity is generated to explore details of the recognition process. The TF . D3h44 in terface is characterised by a high number of polar interactions, including as may as 46 solvent molecules. Conformational changes upon complex formati on are very small and almost exclusively limited to the reorientation of si de-chains. The binding epitope is in complete agreement with earlier mutage nesis experiments. A revaluation of two other antibody-antigen pairs report ed at similar resolutions, shows that all these complexes are very similar with respect to the solvation of the interface, the number of solvent posit ions conserved in the uncomplexed and complexed proteins and the number of water molecules expelled from the surface and replaced by hydrophilic atoms from the binding partner upon complex formation. A strategy is proposed on how to exploit this high resolution structural data to guide the affinity maturation of humanised antibodies. (C) 2001 Academic Press.