Construction of protein binding sites in scaffold structures

We have developed a strategy for grafting a protein-protein interface based on the known crystal structure of a native ligand and receptor proteins in a complex. The key interaction residues at the ligand protein binding inte rface are transferred onto a scaffold protein so that the mutated scaffold protein will bind the receptor protein in the same manner as the ligand pro tein. First, our method identifies key residues and atoms in the ligand pro tein, which strongly interact with the receptor protein. Second, this metho d searches the scaffold protein for combinations of candidate residues, amo ng which the distance between any two candidate residues is similar to that between relevant key interaction residues in the ligand protein. These can didate residues are mutated to key interaction residues in the ligand prote in respectively. The scaffold protein is superposed onto the ligand protein based upon the coordinates of corresponding atoms, which are assumed to st rongly interact with the receptor protein. Complementarity between scaffold and receptor proteins is evaluated. Scaffold proteins with a low superposi ng rms difference and high complementary score are accepted for further ana lysis. Then, the relative position of the scaffold protein is adjusted so t hat the interfaces between the scaffold and receptor proteins have a reason able packing density. Other mutations are also considered to reduce the des olvation energy or bad steric contacts. Finally, the scaffold protein is co minimized with the receptor protein and evaluated. To test the method, the binding interface of barstar, the inhibitor of barnase, was grafted onto sm all proteins. Four scaffold proteins with high complementary scores are acc epted. (C) 2000 John Wiley & Sons, Inc.