Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding.
Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was approximately 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was approximately 10-fold lower. Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.
Gill
R
R
Department of Crystallography, Birkbeck College, London.
Wallach
B
B
Verma
C
C
Ursø
B
B
De Wolf
E
E
Grötzinger
J
J
Murray-Rust
J
J
Pitts
J
J
Wollmer
A
A
De Meyts
P
P
Wood
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003198:Computer Simulation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1