Total alanine-scanning mutagenesis of insulin-like growth factor I (IGF-I) identifies differential binding epitopes for IGFBP-1 and IGFBP-3.
The bioavailability of insulin-like growth factor I (IGF-I) in the serum and tissues is controlled by members of the IGF binding protein family (IGFBP). These proteins form high-affinity complexes with IGF-I and thereby either inhibit or potentiate its mitogenic and metabolic effects. Thus, understanding the IGF-IGFBP interaction at the molecular level is crucial for attempts to modulate IGF-I activity in vivo. We have systematically investigated the binding contribution of each IGF-I amino acid side chain toward IGFBP-1 and IGFBP-3, combining alanine-scanning mutagenesis and monovalent phage display. Surprisingly, most IGF-I residues could be substituted by alanines, resulting in less than 5-fold affinity losses for IGFBP-3. In contrast, binding of IGFBP-1 was more sensitive to alanine substitutions in IGF-I. The glutamate and phenylalanine at positions 3 and 49 were identified as major specificity determinants for IGFBP-1: the corresponding alanine mutations, E3A and F49A, selectively decreased IGFBP-1 binding by 34- and 100-fold, whereas IGFBP-3 affinity was not affected or reduced maximally 4-fold. No side chain specificity determinant was found for IGFBP-3. Instead, our results suggest that the N-terminal backbone region of IGF-I is important for binding to IGFBP-3. The fact that the functional binding epitopes on IGF-I are overlapping but distinct for both binding proteins may be exploited to design binding protein-specific IGF variants.
Dubaquié
Y
Y
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Lowman
H B
HB
D000409:Alanine / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D018970:Insulin-Like Growth Factor Binding Protein 1; D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D007334:Insulin-Like Growth Factor I; D000409:Alanine