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.

Defining the Disulfide Bonds of Insulin-like Growth Factor-binding Protein-5 by Tandem Mass Spectrometry with Electron Transfer Dissociation and Collision-induced Dissociation

“…Our results definitively identify 5 of 9 disulfide bonds in the protein, and determine that the other 4 linkages involve the four cysteines within the conserved GCGCCMTC motif (residues [32][33][34][35][36][37][38][39] in the N-terminal region of the protein.…”

“…As with other IGFBPs, the N-terminal domain (residues 1-84) of IGFBP-5 encodes the primary IGF-binding site, with the C-terminal region (residues 165-252) contributing in a secondary way to binding stability and affinity (34 -36). NMR and protein crystallographic studies of a portion of the N-terminal segment of IGFBP-5 (amino acids 40 -92) have demonstrated that this part of the protein appears to be organized into a tight globular structure that contains an anti-parallel ␤-sheet stabilized by two disulfide bonds linking Cys 47 to Cys 60 and Cys 54 to Cys 80 (34,36). Five residues located near these disulfide-linked cysteines (K68, P69, L70, L73, and L74) have been shown via mutagenesis studies to be major contributors to high affinity binding of IGF-I and IGF-II (34 -37).…”

Binding site for the C‐domain of insulin‐like growth factor (IGF) binding protein‐6 on IGF‐II; implications for inhibition of IGF actions

“…1B). The binding site of IGF-I to mini-IGFBP-5, which is part of the N-domain, is already known [12]. IGF-I and IGF-II share 67% amino acid identity and the residues within the N-domain binding site of IGF-I are completely conserved in IGF-II.…”

“…From these results, two patches of surface-exposed hydrophobic residues that are involved in IGFBP binding were proposed. One patch, including the IGF-II equivalents Glu6, Phe19 and Leu53, was subsequently identified as the N-domain binding site, since the IGF-I/mini-IGFBP-5 crystal structure showed that these side chains were inserted deep into a cleft on mini-BP-5 [12]. The other patch includes the IGF-II equivalents Gly10, Val14, Phe28 and Val43.…”

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) by calpain

“…30-40%), with highest conservation at the N-and C-terminal regions (Figures 6 and 7), which participate in binding to IGFs (Baxter, 2000;Payet et al, 2003;Shand et al, 2003). The structure of the N-terminal domain of IGFBP-5 discloses a rigid, globular fold that consists of a centrally located three-stranded antiparallel b-sheet (Zeslawski et al, 2001). This is in contrast to the central, weakly conserved domain in IGFBPs, for which the NMR spectra presented here indicate a highly flexible, disordered conformation ( Figure 6).…”