Synthesis of the Blood Circulating C-terminal Fragment of Insulin-like Growth Factor (IGF)-binding Protein-4 in Its Native Conformation

Abstract

Insulin-like growth factor-binding proteins play a critical role in a wide variety of important physiological processes. It has been demonstrated that both an Nterminal and a C-terminal fragment of insulin-like growth factor-binding protein-4 exist and accumulate in the circulatory system, these fragments accounting for virtually the whole amino acid sequence of the protein.The circulating C-terminal fragment establishes three disulfide bridges, and the binding pattern of these has recently been defined. Here we show that the monodimensional 1 H NMR spectrum of the C-terminal fragment is typical of a protein with a relatively close packed tertiary structure. This fragment can be produced in its native conformation in Escherichia coli, without the requirement of further refolding procedures, when synthesis is coupled to its secretion from the cell. The recombinant protein crystallizes with the unit cell parameters of a hexagonal system. Furthermore, it binds strongly to heparin, acquiring a well defined oligomeric structure that interacts with insulin-like growth factors, and promotes bone formation in cultures of murine calvariae.

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

“…Limited insights into the three-dimensional organization of IGFBPs have come from results of high-resolution x-ray crystallographic analyses of the isolated N-terminal domain of IGFBP-4 and the C-terminal segments of IGFBP-1 and IGFBP-4 (13,14). One consistent observation from these data is that IGFBPs lack inter-domain disulfide bonds (12)(13)(14)(15). However, as the structure of a fulllength IGFBP has not been solved, possibly because of the dis-ordered nature of the linker segment, this conclusion remains provisional.…”

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

“…There has been a considerable body of work to delineate the determinants of IGFBPs binding to IGFs and vice versa (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(21)(22)(23)(24)(25)(26)(27)(28)(29). The structural information presented in this work is broadly in agreement with these data, but disagrees with reports of a critical role of the completely conserved Gly-187(C) and Gln-193(C) (in the IGFBP4 sequence) for binding of C domains to IGFs (2, 6, 29, and references cited therein): These residues are not in contact with IGF1, although they are close to the IGF1͞CBP4 interface surface.…”

“…However, recent studies indicate that the resulting N-and Cterminal fragments still can inhibit IGF activity and have functional properties that differ from those of the intact proteins (1,3,5,9).…”

“…More recently, low-resolution structures of the C-terminal domain of IGFBP6 (12) and its binding surface on IGF2 (3,12) have been determined with NMR spectroscopy. There is, however, no x-ray structure of a ternary complex of the C-terminal domain of any IGFBPs bound to both the N-terminal domain and IGF, although the C-terminal fragment of IGFBP4 was crystallized recently (9), and also the x-ray structure of the isolated C-terminal fragment of IGFBP1 has been solved (17). We recently reported the x-ray structure of the ternary complex of the N-and C-terminal domains of IGFBP4 bound to IGF1 (10) and described ordered structures for the N-terminal domain of IGFBP4 and IGF1.…”

“…Each IGFBP can be divided into three distinct domains of approximately equal lengths: highly conserved cysteinerich N and C domains and a central linker domain unique to each IGFBP species. Both the N and C domains participate in the binding to IGFs, although the specific roles of each of these domains in IGF binding have not been decisively determined (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The C-terminal domain may be responsible for preferences of IGFBPs for one species of IGF over the other (2,(3)(4)(5)(6)(7)(9)(10)(11)(12)(13); the C-terminal domain is also involved in regulation of the IGF-binding affinity through interaction with extracellular matrix components (1,2,14) and is most probably engaged in mediating IGF1-independent actions (1,4,14).…”