Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues

Biochemistry. 1999 Nov 30;38(48):15863-70. doi: 10.1021/bi9910070.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biological Assay
  • Circular Dichroism
  • Escherichia coli / metabolism
  • Humans
  • Insulin-Like Growth Factor Binding Proteins / chemistry*
  • Insulin-Like Growth Factor I / biosynthesis
  • Insulin-Like Growth Factor I / chemistry*
  • Insulin-Like Growth Factor I / genetics
  • Molecular Sequence Data
  • Molecular Structure
  • Mutation
  • Protein Binding
  • Rats

Substances

  • Insulin-Like Growth Factor Binding Proteins
  • Insulin-Like Growth Factor I