The interaction of insulin-like growth factor-I with the N-terminal domain of IGFBP-5

Abstract

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.

Fig. 1. Sequence and structure alignment (A) of IGFs and single-chain insulin (SCI). Residues that make contacts with mini-IGFBP-5 within 4 Å are highlighted in magenta; residues responsible for binding to IGF-1R are in red and residues in green showed no electron density. (B) Mini-IGFBP-5 with the corresponding N-terminal domains of IGFBP-3, IGFBP-rP1 and IGFBP-rP2; consensus amino acid residues are shown above the sequences; conserved residues are indicated by blue letters. Residues that interact with IGF-I (within 4 Å) are highlighted in magenta. The mini-IGFBP-5 construct had additional Gly and Ser residues from the cloning vector at the N-terminus; residues in green showed no electron density.

Fig. 2. The overall structure of the IGF-I (green)–mini-IGFBP-5 (black) complex. (A) A heavy atom plot. Residues shown in magenta constitute the primary binding sites for interaction with mini-IGFBP-5. Residues in red are determinants for binding to IGF-1R. The first N- and last C-terminal residues are shown in brown and blue, respectively. (B) Interface of the IGF-I–mini-IGFBP-5 complex interactions. Mini-IGFBP-5 is shown as a surface plot (residues in red, negatively charged; blue, positive; white, neutral), IGF is shown in blue. Side chains of the primary binding residues of IGF for mini-IGFBP-5 are shown. (C) Ribbon plot of IGF (green)–mini-IGFBP-5 (grey) with interface residues that form hydrogen bonds highlighted (blue). The interface hydrophobic residues are shown in yellow.

Fig. 3. Stereo figure of the 2F_o – F_c electron density map contoured at 1.0σ over the mean at the mini-IGFBP-5–IGF-I interface. The IGF-I peptide segment is at the left, including the interfacial Phe16, seen here packed against two segments of mini-IGFBP-5, between Val49 (below) and Cys60 (above).