Alanine screening mutagenesis establishes tyrosine 60 of bovine insulin-like growth factor binding protein-2 as a determinant of insulin-like growth factor binding.
The determinants of insulin-like growth factor (IGF) binding to its binding proteins (IGFBPs) are poorly characterized in terms of important residues in the IGFBP molecule. We have previously used tyrosine iodination to implicate Tyr-60 in the IGF-binding site of bovine IGFBP-2 (Hobba, G. D., Forbes, B. E., Parkinson, E. J., Francis, G. L., and Wallace, J. C. (1996) J. Biol. Chem. 271, 30529-30536). In this report, we show that the mutagenic replacement of Tyr-60 with either Ala or Phe reduced the affinity of bIGFBP-2 for IGF-I (4.0- and 8.4-fold, respectively) and for IGF-II (3.5- and 4.0-fold, respectively). Although adjacent residues Val-59, Thr-61, Pro-62, and Arg-63 are well conserved in IGFBP family members, Ala substitution for these residues did not reduce the IGF affinity of bIGFBP-2. Kinetic analysis of the bIGFBP-2 mutants on IGF biosensor chips in the BIAcore instrument revealed that Tyr-60 --> Phe bIGFBP-2 bound to the IGF-I surface 3.0-fold more slowly than bIGFBP-2 and was released 2.6-fold more rapidly than bIGFBP-2. We therefore propose that the hydroxyl group of Tyr-60 participates in a hydrogen bond that is important for the initial complex formation with IGF-I and the stabilization of this complex. In contrast, Tyr-60 --> Ala bIGFBP-2 associated with the IGF-I surface 5.0-fold more rapidly than bIGFBP-2 but exhibited an 18.4-fold more rapid release from this surface compared with bIGFBP-2. Thus both the aromatic nature and the hydrogen bonding potential of the tyrosyl side chain of Tyr-60 are important structural determinants of the IGF-binding site of bIGFBP-2.
Cooperative Research Centre for Tissue Growth and Repair, P. O. Box 10065, Gouger Street, Adelaide, South Australia 5005.
G D
GD
Hobba
A
A
Löthgren
E
E
Holmberg
B E
BE
Forbes
G L
GL
Francis
J C
JC
Wallace
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002417:Cattle; D002942:Circular Dichroism; D006860:Hydrogen Bonding; D018969:Insulin-Like Growth Factor Binding Proteins / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D014443:Tyrosine