The insulin-like growth factor (IGF)binding protein 1 binding epitope on IGF-I probed by heteronuclear NMR spectroscopy and mutational analysis.
NMR spectroscopy studies and biosensor interaction analysis of native and site-directed mutants of insulin-like growth factor I (IGF-I) was applied to identify the involvement of individual residues in IGF-I binding to IGF-binding protein 1 (IGFBP-1). Backbone NMR chemical shifts were found to be affected by IGFBP-1 binding in the following residues: Pro2, Glu3, Cys6, Gly7, Gly19, Pro28-Gly30, Gly32, Arg36, Arg37, Gln40-Gly42, Pro63, Lys65, Pro66, and Lys68-Ala70. Three IGF-I arginine side chains were identified by NMR to participate in IGFBP-1 binding. All IGF-I arginine residues were replaced by alanines, using site-directed mutagenesis, in four single substituted variants, IGF-I(R21A), IGF-I(R50A), IGF-I(R55A), and IGF-I(R56A), and one double replacement mutant, IGF-I(R36A/R37A). Biosensor interaction analysis binding studies demonstrate the involvement of Arg36-Arg37 and Arg50 in IGFBP-1 binding, while experiments with the IGF-I receptor implicate Arg21, Arg36-Arg37, and Arg56 as part of the receptor binding epitope. These overlapping binding surfaces explain why IGF-I receptor and IGFBP-1 binding to IGF-I is competitive. The C terminus of free, but not IGFBP-1-bound, IGF-I is found to exist in two distinct, NMR-detectable conformations at 30 degreesC. One possible explanation for this structural heterogeneity could be cis-trans isomerization of the Cys6-Cys48 disulfide bond.
Jansson
M
M
Department of Structural Chemistry, Pharmacia & Upjohn, SE-11287 Stockholm, Sweden.
Andersson
G
G
Uhlén
M
M
Nilsson
B
B
Kördel
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D004926:Escherichia coli; D014644:Genetic Variation; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry* / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D000939:Epitopes; D018970:Insulin-Like Growth Factor Binding Protein 1; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I