Insulin-like Growth Factor I and Its Binding Proteins:  A Study of the Binding Interface Using B-Domain Analogues

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1−6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His4]-, [Gln9]-, [Lys9]-, [Ser16]-, [Gln9,Ser16]-, and [Lys9,Ser16]IGF-I, that contained substitutions for residues Thr4, Glu9, or Phe16. Substitution of Ser for Phe16 did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe16 is functionally important for IGFBP association. While His4 or Gln9 substitutions had little effect on IGFBP affinity, changing the negative charge of Glu9 to a positive Lys9 selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe16 and adjacent hydrophobic residues (Leu5 and Leu54) form a functional binding pocket for IGFBP association.