Protein loop grafting to construct a variant of tissue-type plasminogen activator that binds platelet integrin alpha IIb beta 3.
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 beta 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 factor-like module of human tissue-type plasminogen activator (t-PA). The resulting variant of t-PA bound to the platelet integrin alpha IIb beta 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 alpha IIb beta 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.
Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
J W
JW
Smith
K
K
Tachias
E L
EL
Madison
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator