Protein Loop Grafting to Construct a Variant of Tissue-type Plasminogen Activator That Binds Platelet Integrin αIIbβ3

Abstract

Protein-protein interactions can be guided by contacts between surface loops within proteins. We therefore investigated the hypothesis that novel protein-protein interactions could be created using a strategy of "loop grafting" in which the amino acid sequence of a biologically active, flexible loop on one protein is used to replace a surface loop present on an unrelated protein.To test this hypothesis we replaced a surface loop within an epidermal growth factor module with the complementarity-determining region of a monoclonal antibody. Specifically, the HCDR3 from Fab-9, an antibody selected to bind the ␤ 3 -integrins with nanomolar affinity (Smith, J. W., Hu, D., Satterthwait, A., Pinz-Sweeney, S., and Barbas, C. F., III (1994) J. Biol. Chem. 269, 32788 -32795), was grafted into the epidermal growth factorlike module of human tissue-type plasminogen activator (t-PA). The resulting variant of t-PA bound to the platelet integrin ␣ IIb ␤ 3 with nanomolar affinity, retained full enzymatic activity, and was stimulated normally by the physiological co-factor fibrin. Binding of the novel variant of t-PA to integrin ␣ IIb ␤ 3 was dependent on the presence of divalent cations and was inhibited by an RGD-containing peptide, demonstrating that, like the donor antibody, the novel t-PA binds specifically to the ligand-binding site of the integrin. These findings suggest that surface loops within protein modules can, at least in some cases, be interchangeable and that phage display can be combined with loop grafting to direct proteins, at high affinity, to selected targets. In principle, these targets could include not only other proteins but also peptides, nucleic acids, carbohydrates, lipids, or even uncharacterized markers of specific cell types, tissues, or viruses.Development of the ability to create novel protein-protein interactions promises to provide important new therapeutic agents as well as unique tools and reagents for the study of key biological processes. Such advances in protein engineering may also provide seminal information about how proteins interact.One strategy for manipulating protein-protein interactions is to employ random or nearly random (e.g. alanine scanning) (1) site-directed mutagenesis to identify amino acid residues critical for binding affinity and specificity. Cumbersome, large scale mutagenesis efforts followed by laborious, time-consuming assays of individual, mutated proteins can sometimes extend this approach to create new molecular interactions. For example, every residue in human growth hormone was scanned for activity prior to re-engineering the molecule to bind the prolactin receptor (2), and a similar strategy facilitated the construction of a variant of interleukin-3 that binds the monomeric ␣ receptor with higher affinity than it binds the interleukin-3 ␣␤ receptor (3). An important current challenge, therefore, is to create more rapid and efficient strategies to engineer proteins with new binding properties. A recent approach that can obviate the need to perform large numbers ...

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