Michelle Schaffer

Rapid Identification of Small Binding Motifs with High-Throughput Phage Display: Discovery of Peptidic Antagonists of IGF-1 Function

A panel of 22 naïve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11). After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX(9)C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.

Phage display-derived ligands provide structural insight into insulin-like growth factor I function

Insulin-like growth factor-I (IGF-I) is a central mediator of cell growth, differentiation and metabolism. Structural characterization of the protein has been hampered by a combination of internal dynamics and self-association that prevent crystallization and produce broad NMR resonances. To better characterize the functions of IGF-I, we have used phage display to identify peptides that antagonize the binding of IGF-I to its plasma binding proteins (IGFBPs) and cell-surface receptor (IGF-R). Interestingly, binding of peptide improves dramatically the quality of the NMR resonances of IGF-I, and enables the use of triple-resonance NMR methods to characterize the complexes. One such peptide, designated IGF-F1-1, has been studied in detail. In the complex, the peptide retains the same loop-helix motif seen in the free state whilst IGF-I contains three helices, as has been seen previously in low-resolution structures in the absence of ligand. The peptide binds at a hydrophobic patch between helix 1 and 3, a site identified previously by mutagenesis as a contact site for IGFBP1. Thus, antagonism of IGFBP1 binding exhibited by the peptide occurs by a simple steric occlusion mechanism. Antagonism of IGF-R binding may also be explained by a similar mechanism if receptor binding occurs by a two-site process, as has been postulated for insulin binding to its receptor. Comparisons with crystallographic structures determined for IGF-I in other complexes suggest that the region around helix 1 of IGF-I is conformationally conserved whereas the region around helix 3 adopts several different ligand-induced conformations. The ligand-induced structural variability of helix 3 appears to be a common feature across the insulin super- family. In the case of IGF-I, exchange between such conformations may be the source of the dynamic nature of free IGF-I, and likely has functional significance for the ability of IGF-I to recognize two signaling receptors and six binding proteins with high affinity.