Insulin-like Growth Factor I and Its Binding Proteins: A Study of the Binding Interface Using B-Domain Analogues ² Barbara A. Magee, ‡ Gary K. Shooter, ‡ John C. Wallace,* ,‡ and Geoffrey L. Francis § CooperatiVe Research Center for Tissue Growth and Repair, Department of Biochemistry, UniVersity of Adelaide, Adelaide, South Australia 5005, Australia, and CooperatiVe Research Center for Tissue Growth and Repair, GroPep Pty. Ltd., P.O. Box 10065BC, Adelaide, South Australia 5000, Australia ReceiVed May 3, 1999; ReVised Manuscript ReceiVed September 7, 1999 ABSTRACT: 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, [His 4 ]-, [Gln 9 ]-, [Lys 9 ]-, [Ser 16 ]-, [Gln 9 ,Ser 16 ]-, and [Lys 9 ,- Ser 16 ]IGF-I, that contained substitutions for residues Thr 4 , Glu 9 , or Phe 16 . Substitution of Ser for Phe 16 did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe 16 is functionally important for IGFBP association. While His 4 or Gln 9 substitutions had little effect on IGFBP affinity, changing the negative charge of Glu 9 to a positive Lys 9 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 Phe 16 and adjacent hydrophobic residues (Leu 5 and Leu 54 ) form a functional binding pocket for IGFBP association. Compact hydrophobic regions are typically found at the interface of protein-protein complexes. These interface sur- faces are composed of complementary hydrophobic deter- minants from both molecules and generally resemble the core of folded proteins (1-3). Functionally important hydrophilic residues are found at the periphery of the hydrophobic interfaces. They contribute to the binding affinity of com- plexed proteins through the formation of salt bridges and hydrogen bonds at the interface surface (2, 4). With these general principles of protein-protein interaction in mind, we examined the surface of insulin-like growth factor I (IGF- I) 1 that is considered important for binding to IGF binding proteins (IGFBPs 1-6) and identified residues that could contribute to the binding surface. We then used directed mutagenesis to study the functional importance of those residues for association with the IGFBPs. IGF-I and IGF-II are anabolic, single-chain polypeptides of 70 and 67 amino acid residues, respectively. Solution NMR studies (5-7) confirm that insulin and the IGFs have similar 3D structures, as predicted from their conserved hydrophobic core residues and an identical arrangement of disulfide bonds. Figure 1 shows the comparative amino acid sequences of human IGF-I, IGF-II, and insulin. Subtle evolutionary changes in the A- and B-domains of the IGFs allow them to associate with the six IGFBPs. In contrast, insulin does not bind the IGFBPs but self-associates to form dimers, tetramers, and hexamers in solution. Details of the IGFBP gene sequences, posttranslational modifications, and known physiological effects can be found in the excellent reviews published by Rechler (8), Kelley et al. (9), and Clemmons (10). The complete tertiary structure is not known for any of the IGFBPs or the IGF-IGFBP complexes; however, previous ² This project was financed through the Australian Government Cooperative Research Centre Scheme. * Corresponding author: Telephone +61-8-8303-5218; Fax +61- 8-8303-4348; E-mail jwallace@biochem.adelaide.edu.au. ‡ University of Adelaide. § GroPep Pty. Ltd. 1 Abbreviations: IGF, insulin-like growth factor; IGFBPs, insulin- like growth factor binding proteins; CD, circular dichroism. FIGURE 1: Comparative amino acid sequences of human insulin, IGF-I, and IGF-II. The underlined residues form helical regions (5-7). 15863 Biochemistry 1999, 38, 15863-15870 10.1021/bi9910070 CCC: $18.00 © 1999 American Chemical Society Published on Web 11/06/1999