High-density mutagenesis by combined DMA shuffling and phage display to assign essential amino acid residues in protein-protein interactions: Application to study structure-function of plasminogen activation inhibitor 1 (PAI-I)

The identification of specific amino acid residues involved in protein-prot ein interaction is fundamental to understanding structure-function relation ships. Supported by mathematical calculations, we designed a high-density m utagenesis procedure for the generation of a mutant library of which a limi ted number of random clones would suffice to exactly localize amino acid re sidues essential for a particular protein-protein interaction. This goal wa s achieved experimentally by consecutive cycles of DNA shuffling, under err or prone conditions, each followed by exposure of the target protein on the surface of phages to screen and select for correctly folded, functional mu tants. To validate the procedure, human plasminogen activator inhibitor 1 ( PAI-1) was chosen, because its 3D structure is known, many experimental too ls are available and it may serve as a model protein for structure-function studies of serine proteinases and their inhibitors (serpins). After five c ycles of DNA shuffling and selection for t-PA binding, analysis of 27 rando mly picked clones revealed that PAI-1 mutants contained an average of 9.1 a mino acid substitutions distributed over 114 different positions, which wer e preferentially located at the surface of the protein. This Limited collec tion of mutant PAI-1 preparations contained multiple mutants defective in b inding to three out of four tested anti-PAI-l monoclonal antibodies. Alignm ent of the nucleotide sequence of defective clones permitted assignment of single dominant amino acid residues for binding to each monoclonal antibody . The importance of these residues was confirmed by testing the properties of single point mutants. From the position of these amino acid residues in the 3D structure of PAI-1 and the effects of the corresponding monoclonal a ntibodies on t-PA-PAI-1 interaction, conclusions can be drawn with respect to this serpin-serine proteinase interaction. (C) 2000 Academic Press.