title: Synthesis of the blood circulating C-terminal fragment of insulin-like growth factor (IGF)-binding protein-4 in its native conformation. Crystallization, heparin and IGF binding, and osteogenic activity.
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 N-terminal 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 1H 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.
authors.affiliation: Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Fernández-Tornero
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Lozano
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.forename: M Angeles
authors.initials: MA
authors.lastname: Jiménez
authors.forename: Ludger
authors.initials: L
authors.lastname: Ständker
authors.forename: Diana
authors.initials: D
authors.lastname: Díaz-Gonzalez
authors.forename: Wolf-Georg
authors.initials: WG
authors.lastname: Forssmann
authors.forename: Pedro
authors.initials: P
authors.lastname: Cuevas
authors.forename: Antonio
authors.initials: A
authors.lastname: Romero
authors.forename: Guillermo
authors.initials: G
authors.lastname: Giménez-Gallego
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001846:Bone Development; D001842:Bone and Bones / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism; D006493:Heparin / Q000737:chemistry*; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000097:blood* / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D004220:Disulfides; D018974:Insulin-Like Growth Factor Binding Protein 4; D006493:Heparin