Structural Origins of the Functional Divergence of Human Insulin-Like Growth Factor-I and Insulin ²,‡ Andrzej M. Brzozowski,* Eleanor J. Dodson, Guy G. Dodson, Garib N. Murshudov, Chandra Verma, Johan P. Turkenburg, Frederik M. de Bree, # and Zbigniew Dauter § Structural Biology Laboratory, Chemistry Department, UniVersity of York, Heslington, York YO10 5DD, United Kingdom ReceiVed January 28, 2002; ReVised Manuscript ReceiVed April 23, 2002 ABSTRACT: Human insulin-like growth factors I and II (hIGF-I, hIGF-II) are potent stimulators of cell and growth processes. They display high sequence similarity to both the A and B chains of insulin but contain an additional connecting C-domain, which reflects their secretion without specific packaging or precursor conversion. IGFs also have an extension at the C-terminus known as the D-domain. This paper describes four homologous hIGF-1 structures, obtained from crystals grown in the presence of the detergent SB12, which reveal additional detail in the C- and D-domains. Two different detergent binding modes observed in the crystals may reflect different hIGF-I biological properties such as the interaction with IGF binding proteins and self-aggregation. While the helical core of hIGF-I is very similar to that in insulin, there are distinct differences in the region of hIGF-I corresponding to the insulin B chain C-terminus, residues B25-B30. In hIGF-I, these residues (24-29) and the following C-domain form an extensive loop protruding 20 Å from the core, which results in a substantially different conformation for the receptor binding epitope in hIGF-I compared to insulin. One notable feature of the structures presented here is demonstration of peptide-bond cleavage between Ser35 and Arg36 resulting in an apparent gap between residues 35 and 39. The equivalent region of proinsulin is involved in hormone processing demanding a reassessment of the structural integrity of hIGF-I in relation to its biological function. Human insulin-like growth factor I (hIGF-I) is a 70-amino acid single chain protein that mediates somatic growth. It has a high (45-52%) sequence similarity with the B and A chains of human insulin, and 67% sequence identity with human insulin-like growth factor-II (IGF-II) (see Figure 1) (1). A number of NMR studies (2-5) and a few recent crystallographic analyses (6, 7) have revealed the essentially identical nature of the core region and the organization of the three helical segments of insulin and hIGF-I. The short N- and C-terminal extensions in hIGFs are directed away from the body of the molecule and are generally mobile. These regions are not detected by NMR and are only partially visible in the crystallographic analyses. Although the IGF- specific C-domain is covalently attached to the region that is the equivalent of the A and B chains of insulin, it is still relatively mobile. The presence of the C-domain does not affect the conformation of the A chain, which is insulin- like. It is, however, associated with structural differences in ² This work was partially supported by the European Community (Human Capital and Mobility Program, Contract No. CHRX-CT94- 0556). The infrastructure of the Structural Biology Laboratory at York is supported by the BBSRC. We thank the European Union for support of the work carried out at EMBL Hamburg outstation through the HCMP access to large installations project (Contract No. CHGE-CT93- 0040). ‡ Atomic coordinates of the hIGF-I-esrf, hIGF-I-dares, hIGF-I-hamb- RT, hIGF-I-inh-RT crystal structures have been deposited in Protein Data Bank (accession codes: 1GZR, 1H02, 1GZZ, 1GZY). * Corresponding author. E-mail: marek@ysbl.york.ac.uk; tele- phone: +44-1904-432570; fax: +44-1904-410519. # Present address: Netherlands Institute for Brain Research, Aca- demic Medical Center, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. § Present address: Synchrotron Radiation Research Section, NCI, Brookhaven National Laboratory, Bldg. 725A-X9, Upton, NY 11973, USA. 1 Abbreviations: hIGF, insulin-like growth factor; IGFBP, IGF- binding protein; IGF-1(2)R, IGF-type 1 and type 2 receptor; IR, insulin receptor; ELISA, enzyme-linked immunosorbent assay; TRIS, (Tris- [hydroxymethyl]aminomethane); SB12, N-dodecyl-N,N-dimethyl-3- ammonio-1-propanesulfonate; big deoxy CHAPS, N,N-bis(3-D-glu- conoamidopropyl) deoxycholamide; 2K PEG MME, poly(ethylene glycol) monomethyl ether of 2000 M.W. FIGURE 1: The sequence and chain organization of hIGF-I, hIGF- II, and insulin. The IGF-specific C- and D-domains are colored grey and pink, respectively; the B and A chains of insulin and their equivalents in hIGF-I/II are highlighted in yellow and blue, respectively. Residues important for the IGF-1R or IR binding are in red, with residues responsible for association with IGFBPs in green (mutations of the highlighted residues result in a minimum 90% drop in binding; residues for which substitution results in even higher impact on affinities toward receptors and IGFBPs are in italic (for details, see refs 8-11). 9389 Biochemistry 2002, 41, 9389-9397 10.1021/bi020084j CCC: $22.00 © 2002 American Chemical Society Published on Web 07/04/2002