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