Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Binding Sites
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Circular Dichroism
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Crystallography, X-Ray
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GTP-Binding Proteins / chemistry*
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GTP-Binding Proteins / metabolism*
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Guanylyl Imidodiphosphate / metabolism
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Humans
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Kinetics
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Point Mutation
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Protein Conformation*
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Protein Serine-Threonine Kinases / chemistry*
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Protein Serine-Threonine Kinases / metabolism*
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Proto-Oncogene Proteins / chemistry*
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-raf
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Sequence Homology, Amino Acid
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Signal Transduction
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rap GTP-Binding Proteins
Substances
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Proto-Oncogene Proteins
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Guanylyl Imidodiphosphate
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-raf
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GTP-Binding Proteins
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rap GTP-Binding Proteins