Abstract
Functional regulation via conformational dynamics is well known in structured proteins, but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (h IGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of h IGFBP2, the linker domain (L-h IGFBP2) retains its intrinsic disorder upon binding IGF-1 but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.
Introduction
The insulin-like growth factor (IGF) system plays an essential role in cell growth, differentiation, and function, and in recent years has become an important target for cancer therapeutics, with more than 30 anti-cancer drugs focusing on this system. This system consists of two peptide hormones: IGF-1 and -2; the receptors: IGF-1R and IGF-2R; six soluble, high-affinity IGF-binding proteins (IGFBPs; numbered 1-6) and IGFBP proteases (Fig. 1 ). IGF-1 and -2 are small proteins (~7.5 kDa) that circulate in the bloodstream and function in signaling by binding to IGF receptors. The biological activities of the IGFs are modulated by IGFBPs, which bind IGFs with higher affinities than the IGF-1R, thereby restricting their delivery to the IGF-1R. Proteolysis of the IGFBPs dissociates IGFs from the complex, enabling them to bind to and activate their receptors (Fig. 1 ).
The biological actions of the IGF: IGFBP: IGF-R axis has been studied extensively. However, a comprehensive understanding of the structural basis for IGF-IGFBP interactions is still lacking. The IGFBPs consist of structured globular N- and C-domains joined by a central (linker) domain. The three-dimensional structures of full-length IGFBPs have not yet been determined, although structures are available for the N- and C- terminal domains of the different IGFBPs either free in solution or bound to IGFs. These studies have shown that all IGFBPs interact similarly with IGFs and that binding sites for the IGFs are located primarily in the N-terminal and C-terminal domains of IGFBPs. The intrinsically disordered linker domain, which contains several post-translational modification motifs and the IGFBP-protease cleavage sites, has been proposed to merely tether the N- and C-terminal domains. However, deletion of the linker domain from full-length proteins results in the loss of IGF binding affinity, indicating that this domain may contribute to IGF-binding in the full-length proteins. Further, in some of the IGFBPs, proteolysis in the linker domain requires the binding of IGFs. Despite its apparent importance in IGFBP function, the structure of the linker domain and its interaction with IGFs have not been characterized. Here we have investigated the interaction of the linker domain of human IGFBP2 (L-h IGFBP2; residues A97-C191) with IGFs using NMR spectroscopy and surface plasmon resonance (SPR). Our study confirms that L-h IGFBP2 is intrinsically disordered and shows that it retains moderate binding affinity to IGFs (K d ~ 4 µM). IGF binding has a specific effect on the dynamics of L-h IGFBP2 residues that interact with IGF-1, but also on the protease cleavage sites. This altered dynamics may explain the IGF-dependent proteolysis of IGFBP2 in this domain. Our understanding of the role of dynamics in the functional regulation of the IGF-system offers new insights that will help guide the design and development of IGFBP- based analogs for inhibiting IGF-IGF-1R signaling and growth of IGF-dependent tumors.
Results