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, [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.
Magee
B A
BA
Cooperative Research Center for Tissue Growth and Repair, Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5005, Australia.
Shooter
G K
GK
Wallace
J C
JC
Francis
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000737:chemistry*; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D051381:Rats
D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I