Quantitative structure-activity analysis correlating Ras/Raf interaction in vitro to Raf activation in vivo.
Binding of Ras to c-Raf-1 is a pivotal step of many mitogenic signalling pathways. Based on the recent crystal structure of the complex of Rap1A with the Ras-binding domain of Raf, mutations were introduced in c-Raf-1 and their effects on Ras/Raf binding affinity in vitro and Ras/Raf regulated gene expression in vivo were analysed. Our data reveal an empirical semilogarithmic correlation between dissociation constants and Raf-induced gene activity. The functional epitope that primarily determines binding affinity consists of residues Gln 66, Lys 84 and Arg 89 in Raf. This quantitative structure-activity investigation may provide a general approach to correlate structure-guided biochemical analysis with biological function of protein-protein interactions.
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Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddamm, Federal Republic of Germany.
Janknecht
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Herrmann
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Nassar
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Wittinghofer
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020668:rap GTP-Binding Proteins
D011518:Proto-Oncogene Proteins; D006165:Guanylyl Imidodiphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins