Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding

Raj Gill1, Chandra Verma2, Brenda Wallach3, Birgitte Ursø3, Jim Pitts4, Axel Wollmer5, Pierre De Meyts3 and Steve Wood1,6

1 Department of Biochemistry, School of Biological Sciences, University of Southampton, 6 Bassett Crescent East, Southampton SO16 7PX, 2 Department of Chemistry, University of York, Heslington, York YO1 5DD, UK, 3 Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark, 4 Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK and 5 Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Klinikum, Pauwelsstrasse 30, D-52057 Aachen, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18–61,6–48,47–52] and IGF-1 swap [18–61,6–47, 48–52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a ~30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was ~200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region {alpha}-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region {alpha}-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.

Keywords: insulin/insulin-like growth factor-1/molecular dynamics/receptor binding/structure–function relationships


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF-1) is a 70 amino acid single-chain protein which has many growth-promoting and metabolic activities (for a review, see Froesch et al., 1985Go). The first 29 residues of IGF-1 are homologous with the B-chain of insulin (B-region; 1–29). The following 12 residues are analogous to the C-peptide of proinsulin (C-region; 30–41) and the next 21 residues are homologous to the A-chain of insulin (A-region; 42–62). The carboxy-terminal octapeptide (D-region; 63–70) has no counterpart in the insulin molecule. In the absence of a crystal structure, the tertiary structure of IGF-1 has been modelled on that of porcine insulin (see Figure 10aGo; Blundell et al., 1978Go, 1983Go). 2-D NMR studies have confirmed that the solution structure of IGF-1 is consistent with this model (Cooke et al., 1991Go; Sato et al., 1993Go). In the insulin fold an A-chain of 21 residues and a B chain of 30 residues are cross-linked by two disulphide bridges (A20–B19 and A7–B7) on either side of the B-chain {alpha}-helix. A third intrachain disulphide (A6–A11) bridges the loop between the two short anti-parallel A-chain helices (see Figure 9aGo; Baker et al., 1988Go). The corresponding disulphide connectivity [18–61,6–48,47–52] has been confirmed for plasma-derived human IGF-1 (Axelsson et al., 1992Go).




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Fig. 10. (a) The energy-minimized IGF-1 model. The B- and D-regions are in light grey, the C-region is in dark grey and the A-region is in white. The side chains of the insulin receptor binding determinants (Tyr24 and Val44) and the IGF-1 receptor binding determinants (Tyr24 and Tyr31) are also shown.( b) The model of IGF-1 swap. The structure was generated from the IGF-1 model by changing the disulphide connectivities and subjecting the energy-minimized structure to a 1.0 ns high-temperature molecular dynamics simulation. The disulphide dihedral angles are as follows: 18–61, –83°; 6–47, +97°; 48–52, +97° (net dihedral: +118°).

 



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Fig. 9. (a) The X-ray structure of insulin (entry 4ins in the Brookhaven database) depicted using MOLSCRIPT (Kraulis, 1991Go). The B-chain is in light grey and the A-chain is in white. The side chains of the insulin receptor binding determinants (B25 Phe and A3 Val) are also shown. The disulphide dihedral angles are as follows: 18–61, –93°; 6–48, +93°; 47–52, +97° (net dihedral: +97°). (b) The NMR structure of insulin swap (entry 1xgl in the Brookhaven database).

 
Unexpectedly, oxidative refolding of human IGF-1 expressed in Escherichia coli yields not only the native molecule but also a similar quantity of a disulphide-swapped isomer which elutes earlier upon reversed-phase chromatography (Meng et al., 1988Go). The disulphide connectivity of the isomer has been determined to be [18–61,6–47,48–52] (Iwai et al., 1989Go). Interestingly, both disulphide isomers of IGF-1 are also secreted in similar quantities from E.coli (Forsberg et al., 1990Go) and Saccharomyces cerevisiae (Elliot et al., 1990Go). Accordingly, the thermodynamic stabilities of native IGF-1 and IGF-1 swap with respect to the denatured state have been shown to be similar (Miller et al., 1993Go). Naturally, the existence of two distinct tertiary structures arising from one primary sequence is unexpected (Anfinsen, 1973Go). IGF-1 swap has a lower affinity than native IGF-1 for the human placental IGF-1 receptor. Quantitatively, there are conflicting values of the binding constant in the literature, ranging from no affinity (Tamura et al., 1988Go; Elliot et al., 1990Go; Forsberg et al., 1990Go) to a 5–10-fold reduction in affinity (Meng et al., 1988Go; Axelsson et al., 1992Go; Miller et al., 1993Go).

The IGF-1 and insulin receptors are membrane glycoproteins composed of two extracellular {alpha}-subunits and two transmembrane ß-subunits linked by disulphide bonds to give an ({alpha}ß)2 dimer (Massagué and Czech, 1982Go; Ullrich et al., 1986Go). Receptor occupation by ligand modulates phosphorylation events on the cytoplasmic domains that initiate recognition and activation of signal transduction mechanisms in the cell. Evidence has accumulated to indicate that multiple conformational perturbations may be necessary to induce high-affinity binding of insulin to its receptor. Insulin binding to its receptor does not conform to the law of mass action due to the phenomenon of negative cooperativity, whereby initial insulin-receptor binding reduces the affinity of the vacant receptor pool for subsequent insulin molecules (De Meyts et al., 1973Go). There is no evidence that multiple conformational perturbations are necessary for high-affinity binding of IGF-1 to the IGF-1 receptor, although this system has also been shown to exhibit negative cooperativity (Christoffersen et al., 1994Go). The growth-promoting effects of insulin and the metabolic activity of IGF-1 are thought to arise from cross-binding to each other's receptors.

In this paper, we describe the expression and purification of the two disulphide isomers of human IGF-1 secreted from yeast. Isocratic reversed-phase chromatography was employed to ensure complete separation of the isomers. We present equilibrium and kinetic binding data at the human IGF-1 receptor overexpressed at the surface of stably transfected baby hamster kidney cells. Both disulphide isomers were also assayed for their ability to stimulate thymidine uptake in NIH 3T3 cells (via the IGF-1 receptor) and glucose uptake in rat fat cells (via the insulin receptor). The isomers were structurally characterized by circular dichroism (CD) spectroscopy. We present the results of a 1 ns trajectory of molecular dynamics simulation that explores the conformational space accessible to the swapped disulphide isomer of IGF-1. We demonstrate the relationship of the IGF-1 swap model to the CD spectra and discuss the relative biological properties of the isomers in terms of our current understanding of the structure–function relationships of insulin and IGF-1.


    Materials and methods
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 Materials and methods
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 References
 
Cloning, expression and purification of IGF-1 in yeast

Cloning, expression and purification of human IGF-1 from S.cerevisiae has been described (Gill et al., 1996Go). An extinction coefficient of 0.69 OD280 units/cm was obtained for a 1 mg/ml solution of monomeric IGF-1 in 10% acetic acid.

Separation of IGF-1 disulphide isomers

Disulphide isomers of IGF-1 were separated by reversed-phase high-performance liquid chromatography (RP-HPLC). A 1 mg amount of monomeric IGF-1 was dissolved in 100 mM sodium phosphate, pH 2.0, and loaded on to a Vydac 218 TP 54 analytical-scale column (0.46x25 cm, butyl-derivatized end-capped 5 µm silica with 300 Å pores; Separations Group, Hesperia, CA). An isocratic separation in a phosphate buffer–acetonitrile system was carried out in order to resolve fully both IGF-1 species. At 74% solvent A (100 mM sodium phosphate, pH 2.0) and 26% solvent B (acetonitrile), wild-type IGF-1 resolved into two isoforms. Each fully resolved disulphide isomer was collected and desalted by gradient RP-HPLC using an acetonitrile–water–propan-2-ol–TFA gradient (Miller et al., 1993Go). The amino acid composition of each IGF-1 isomer was verified by amino acid analysis as described previously (Gill et al., 1996Go).

SDS–PAGE

SDS–PAGE of both IGF-1 disulphide isomers was performed on a Phast system apparatus (Pharmacia) using 20% homogeneous gels. Recombinant native human IGF-1 produced in E.coli (Celltrix) was run as a control. Protein was visualized by Coomassie Brilliant Blue staining.

Equilibrium binding to the IGF-1 receptor

The relative binding affinities of the native and disulphide-swapped folds of wild-type IGF-1 for the IGF-1 receptor were determined by a competition displacement assay using receptors overexpressed on the surface of stably transfected baby hamster kidney cells (Andersen et al., 1992Go; Gill et al., 1996Go). The binding data (percentage bound, duplicate determination) was fitted to a model assuming one high-affinity bound ligand per receptor dimer (Christoffersen et al., 1994Go) from which the affinity (KD) was calculated.

Dose–response of negative cooperativity at the IGF-1 receptor

Dissociation of [125I]IGF-1 from the IGF-1 receptor was allowed to proceed for 30 min at 4°C in a 40-fold dilution in the presence of increasing concentrations of unlabelled wild-type IGF-1 and IGF-1 swap as described previously (Christoffersen et al., 1994Go). The amount of bound tracer at the different cold ligand concentrations was normalized with respect to the amount of bound tracer after dilution alone and plotted against the cold ligand concentration.

Thymidine incorporation assay and fat cell assay

Native and disulphide-swapped IGF-1 were assayed for thymidine incorporation in NIH 3T3 cells and lipogenesis from glucose in rat fat cells as described previously (Gill et al., 1996Go).

CD spectroscopy

CD spectra of 0.92 mg/ml solutions of native IGF-1 and IGF-1 swap were recorded in 5% (v/v) acetic acid as described previously (Gill et al., 1996Go). For comparison purposes, protein concentrations of the native and disulphide-swapped folds were normalized for tyrosine content (Balestrieri et al., 1978Go). The far-UV CD spectra were analysed with respect to secondary structural composition using the CONTIN program (Provencher and Glöckner, 1981Go).

Computer modelling and molecular dynamics of disulphide-swapped wild-type IGF-1

The wild-type IGF-1 model (Blundell et al., 1983Go) was used as the starting structure. The structure was modelled using the CHARMM empirical energy function (Brooks et al., 1983Go) with the TIP3P model (Jorgensen et al., 1983Go) for water and the parameter set 19 (Neria et al., 1996Go) for the protein. The structure was solvated with a shell of water molecules 6 Å thick using the program SOLVATE kindly provided by Professor Grubmuller (Grubmuller, unpublished data). A shell of water molecules was used instead of a large sphere of water molecules as it has been found to reproduce the essential dynamical properties of the latter solvation scheme (Steinbach and Brooks, 1993Go; Ma and Karplus, 1997Go) with large reductions in computational time. Positions of hydrogen atoms attached to the polar atoms were built using the HBUILD (Brünger and Karplus, 1988Go) module of CHARMM. The charge states of the acidic groups (Asp/Glu/C-terminal) were set to zero to correspond to the low-pH experimental data. Non-bonded interactions were truncated at 12 Å by shifting the electrostatics (Brooks et al., 1983Go). The structure was subjected to a protocol of energy minimizations with decremental constraints (Bruccoleri and Karplus, 1986). The disulphide-swapped structure was generated from the minimized native IGF-1 structure by changing the disulphide connectivities. This disulphide-swapped structure was subjected to the same protocol of minimizations as for the native IGF-1 structure. Both minimized structures were subjected to 1.0 ns molecular dynamics simulations by coupling them to a heat bath at 300 K through a coupling constant of 1.0 ps (Berendsen et al., 1984Go). SHAKE (Ryckaert et al., 1977Go; van Gunsteren and Karplus, 1982Go) was used to constrain the vibrations of bonds, thereby allowing a 2.0 fs time step in the integrations of the equations of motion. Structures were taken at 100 ps intervals and subjected to steepest descent and adopted basis Newton–Raphson minimizations until the gradient of the potential was <=10–2 kcal/mol.Å. However, the resulting structure of IGF-1 swap remained close to the starting structure. Consequently, a high-temperature molecular dynamics simulation was carried out after removing the added solvent. A series of 1.0 ns molecular dynamics simulations at high temperatures followed by energy minimizations with the same criteria as outlined above were employed on the published NMR structures of insulin (native and swap) and were extended to the IGF-1 swap model.


    Results
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Folded recombinant human IGF-1 was secreted from yeast at 1 mg/l as determined by radioimmunoassay. Purification of ~10 mg of immunoreactive IGF-1 from a 10 l fermentation resulted in ~1.25 mg of monomer, the majority of the immunoreactive IGF-1 being in the form of disulphide cross-linked aggregates. Conventional gradient reversed-phase chromatography of the monomeric IGF-1 was unable to resolve fully the two disulphide isomers, necessitating the use of an isocratic separation in phosphate buffer followed by desalting by gradient reversed-phase chromatography (Figure 1Go). RP-HPLC of 1 mg of monomeric IGF-1 yielded 125 µg of native IGF-1 and 75 µg of IGF-1 swap (a ratio of 5:3). These yields from the 10 l fermentation were sufficient for biochemical studies but four further 10 l fermentations were required to obtain sufficient material for CD studies. SDS–PAGE of the two IGF-1 disulphide isomers demonstrated that when denatured, both molecules co-migrate with a native human IGF-1 standard produced in E.coli (Figure 2Go). The yeast-produced material also shows extra bands running at a slightly larger size than the standard. The MALDI-TOF mass spectrum displays a peak at 7666 Da (expected mass 7670 Da; 0.05% error) and another at 7974 Da (4.02% difference) (data not shown). This 308 Da difference compares well with the mass of two mannose residues (4.5% difference) found attached to Thr29 in approximately half of the IGF-1 molecules expressed in yeast (Gellerfors et al., 1989Go).



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Fig. 1. RP-HPLC of monomeric wild-type IGF-1 purified by gel filtration. (a) 1 mg of IGF-1 partially resolved into disulphide isomers by the gradient of Miller et al. (1993). (b) 1 mg of IGF-1 chromatographed using an isocratic phosphate buffer–acetonitrile system. (c) Desalting of isocratically resolved disulphide isomers using the gradient of Miller et al. (1993). Final yields: IGF-1 swap (peak 1), 75 µg; native IGF-1 (peak 2), 125 µg.

 


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Fig. 2. SDS–PAGE analysis of wild-type IGF-1 disulphide isomers and an IGF-1 standard produced in E.coli. 1 µg samples were denatured in SDS and ß-mercaptoethanol and electrophoresed on a 20% homogeneous Phast-Gel (Pharmacia). Lanes 1 and 5, low molecular weight protein ladder; lane 2, native IGF-1 standard from E.coli; lane 3, yeast secreted IGF-1 swap (peak 1); lane 4, yeast-secreted native IGF-1 (peak 2). Protein was visualized by Coomassie Brilliant Blue staining; 20 ng of protein can be visualized by this method, implying that any impurities must be present at below 2% (w/w).

 
Competition data for the inhibition of [125I]IGF-1 binding to BHK cells overexpressing the IGF-1 receptor by native IGF-1 and IGF-1 swap are presented in Figure 3Go. The concentration of native IGF-1 required for half-maximum inhibition of tracer binding was ~1 nM. The corresponding value for IGF-1 swap was ~30 nM, a reduction of ~30-fold in affinity. IGF-1 displayed full negative cooperativity at the IGF-1 receptor (Figure 4Go). Although IGF-1 swap did not display full negative cooperativity at the highest dose tested, from the parallel slopes of the linear drop regions of both sigmoid curves, the concentration of IGF-1 swap required to elicit the same dissociation enhancing effect as native IGF-1 was ~30 fold higher, in line with its loss in IGF-1 receptor affinity. Native IGF-1 was capable of stimulating a fivefold increase in DNA synthesis (above basal) in NIH 3T3 cells overexpressing the IGF-1 receptor, with an ED50 of ~0.4 nM (Figure 5Go). At the concentrations assayed, swapping of the disulphides resulted in a ~40 fold decrease in mitogenicity. In the lipogenesis assay, the potency of native IGF-1 at the insulin receptor was ~400-fold lower than that of insulin and swapping of the disulphides further reduced the potency by at least 200-fold (Figure 6Go).



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Fig. 3. Competition curves for the inhibition of [125I]IGF-1 binding to the IGF-1 receptor on stably transfected baby hamster kidney cells by increasing concentrations of native IGF-1 and IGF-1 swap (averaged duplicate points fitted to a receptor dimer model).

 


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Fig. 4. Dose–response relationships for the acceleration of dissociation of [125I]IGF-1 from the IGF-1 receptor on stably transfected baby hamster kidney cells by increasing concentrations of native IGF-1 and IGF-1 swap (average of three experiments).

 


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Fig. 5. [3H]Thymidine uptake by serum-starved NIH 3T3 fibroblasts after 1 h exposure to increasing concentrations of native IGF-1 and IGF-1 swap (average of three experiments).

 


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Fig. 6. Stimulation of lipogenesis in isolated primary rat adipocytes after 2 h incubation with increasing concentrations of native IGF-1 and IGF-1 swap (averaged triplicate points).

 
The superposed far-UV CD spectra of native and disulphide-swapped IGF-1 are presented in Figure 7Go. To determine the secondary structural composition, the spectra were analysed with the program CONTIN (Provencher and Glöckner, 1981Go) using sets of reference spectra. The analysis of {alpha}-helical content is 30% for native IGF-1 and 25% for IGF-1 swap. In the near-UV (Figure 8Go), the ellipticity at 250 nm is more positive for IGF-1 swap than for native IGF-1. Computer modelling of the disulphide swap followed by high-temperature molecular dynamics simulations resulted in uncoiling of the first A-region {alpha}-helix accompanied by a change in the relative orientation of the A- and B-regions (Figure 10bGo), a conformational change analogous to that demonstrated upon swapping of the disulphides in insulin (Figure 9bGo).



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Fig. 7. Superposed far-UV CD spectra of native IGF-1 and IGF-1 swap in 5% acetic acid. Protein concentrations were normalized for tyrosine content.

 


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Fig. 8. Superposed near-UV CD spectra of native IGF-1 and IGF-1 swap in 5% acetic acid. Protein concentrations were normalized for tyrosine content.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Binding of IGF-1 swap to the human placental IGF-1 receptor has been previously characterized but there are conflicting values of the binding constant in the literature. The discrepancies may arise from the incomplete separation of the disulphide isomers during purification or from cross-binding to the insulin receptor and insulin–IGF-1 hybrid receptors on the placental membrane (Kasuya et al., 1993Go). IGF-1 swap had a ~30 fold loss in affinity for the IGF-1 receptor compared with native IGF-1 but, once bound, was capable of inducing negative cooperativity with a dose–response curve parallel to that of native IGF-1, albeit at doses ~30 fold higher. The parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was ~200-fold lower than that of native IGF-1. The binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor.

The far-UV CD spectrum of native IGF-1 is similar to that of insulin (Wood et al., 1975Go) with an intense positive band at 196 nm and two negative bands at 207 and 222 nm, characteristic of peptide bonds arranged in a right-handed helical array. The far-UV CD spectrum of wild-type IGF-1 swap shows a reduction in {alpha}-helical content compared with native wild-type IGF-1. The analysis of the {alpha}-helical content of native IGF-1 (30%) agrees with that expected from the structure of IGF-1 in which residues 8–18, 43–47 and 54–58 are in a helical conformation (31.5%). The {alpha}-helical content of IGF-1 swap (25%) indicates that three or four out of IGF-1's 70 residues have undergone a helix to coil transition.

In the near-UV where the CD is due to transitions of the aromatic and disulphide side chains, the spectrum of native IGF-1 is qualitatively similar to that of insulin (Wollmer et al., 1980Go). The work of Linderberg and Michl (1970) demonstrates that the sign of the 250 nm band obeys a quadrant rule, i.e. the sign for a right-handed disulphide with a dihedral angle <90° is negative, whereas the sign for a left-handed disulphide with a dihedral angle >90° is positive. Accordingly, the near-UV spectrum of insulin which contains one right-handed disulphide (A20–B19) and two left-handed disulphides (A7–B7, A6–A11) possesses a positive 250 nm band arising from the net left-handed contribution. The increased ellipticity at 250 nm in the spectrum of IGF-1 swap compared with native IGF-1 indicates a greater net dihedral angle for the left-handed disulphides in IGF-1 swap than for those in native IGF-1.

In the absence of a structure for IGF-1 swap it is impossible to interpret the biological and biophysical data. The 1H NMR spectrum of IGF-1 swap displays substantial differences in secondary shifts when compared with that of native IGF-1 and structure solution has been hampered by severe resonance overlap (Miller et al., 1993Go). However, the solution structure of insulin swap (a metastable disulphide isomer generated by thiol exchange) has been successfully determined by 2D-NMR (Hua et al., 1995Go). Comparison of insulin swap with native insulin (Figure 9aGo) reveals uncoiling of the first A-chain {alpha}-helix and a rearrangement in the orientation of the A and B chains by ~30° (Figure 9bGo). Recent NMR studies of IGF-1 swap indicate that its structure is similar to that of insulin swap (Kobayashi, 1995Go)

In order to interpret the biological and biophysical data on IGF-1 swap in the absence of an NMR structure, it was decided to model its structure. The disulphides of native IGF-1 (Figure 10aGo) were swapped and initial energy minimizations resulted in a structure close to the native structure (the root mean square difference between the two stuctures was 0.9 Å over C{alpha} atoms) with differences limited to the vicinity of the swapped disulphides. High-temperature molecular dynamics can be employed to explore alternative conformations of proteins, particularly those involving large conformational rearrangements (Elamrani et al., 1996Go; Kazmirski and Dagget., 1998Go). To validate this methodology, a series of 1.0 ns molecular dynamics simulations, followed by energy minimizations, were carried out on a disulphide-swapped model of insulin generated from native insulin in a manner analogous to that used for IGF-1. We found that at 400°C, the structural rearrangements observed in the NMR structure of insulin swap were qualitatively reproduced (C.Verma and R.Gill, unpublished data). This success prompted us to employ the same methodology to model IGF-1 swap; however, in this case uncoiling of the first A-region helix was observed at 550°C The higher temperature required for IGF-1 swap can be attributed to the first A-region helix being part of a long segment of polypeptide chain and hence more constrained than the corresponding helix in insulin which is N-terminal. During the molecular dynamics of IGF-1 swap the first A-region helix completely uncoiled by 300 ps, with the partial uncoiling in the first 200 ps being accompanied by a correlated change in the orientation of the second A-region helix by about 30° (Figure 10bGo).

Just how valid is the model of IGF-1 swap and what does it tell us about its structure–function relationships? The uncoiling of the first five residues of the first A-region {alpha}-helix results in a total {alpha}-helical content of 23%, which compares well with the 25% predicted from the far-UV CD spectrum. The increase in the net disulphide dihedral angle of 21° is consistent with the increase in the positive 250 nm band in the near-UV CD spectrum. The global rearrangement in the orientation of the A- and B-regions is similar to that demonstrated for insulin swap (Figure 9bGo); the side chain packing of Ile43 (A2 Ile) and Tyr60 (A19 Tyr) is maintained despite the loss of {alpha}-helical structure and contributes to a reordered hydrophobic core.

Assuming that the model of IGF-1 swap correctly reflects its true structure, how can we account for its receptor binding properties in terms of our present understanding of how insulin and IGF-1 bind to their cognate receptors? Receptor recognition by insulin appears to be a multipoint attachment requiring population of a conformer unlike that seen in X-ray and NMR structures of the native protein. Specifically, initial binding of the insulin B25 Phe side chain with a hydrophobic insulin receptor pocket is thought to be accompanied by a movement of the CO2H terminal B-chain residues away from the insulin core and an adjustment of the underlying NH2 terminal A-chain residues away from an {alpha}-helical conformation to orientate A3 Val for interaction with a second receptor pocket (Nakagawa and Tager, 1992Go). IGF-1 is also thought to bind initially to the IGF-1 receptor via the homologous Tyr24 but the interaction is stabilized by binding of Tyr31 in the C-region to an IGF-1 receptor pocket without counterpart in the insulin receptor (De Meyts, 1994Go). As for insulin and IGF-1 binding to each others receptors, specificity must be determined by the structural differences between the two ligands and within the common binding sites of the two receptors (Kjeldsen et al., 1991Go). The absence of a Tyr31 equivalent in insulin is thought to reduce its affinity for the IGF-1 receptor whereas the presence of the bulky C-region is thought to reduce the affinity of IGF-1 for the insulin receptor, either by steric effects and/or by preventing the conformational change necessary for high affinity binding (Cascieri and Bayne, 1990Go).

The small reduction in activity of IGF-1 swap (~30-fold lower than that of native IGF-1) implies that the detailed configuration of the IGF-1 receptor binding surface is accessible from the disulphide-swapped conformation. From the IGF-1 swap model it is apparent that Tyr24 and Tyr31 could bind their respective receptor pockets without too much difficulty. Given the similarity between the structure of insulin swap and the model of IGF-1 swap presented here, the detailed configuration of the insulin receptor binding surface (involving Tyr24 and Val44) should also be equally accessible from both structures. Consequently, it is intriguing that although the insulin activity of insulin swap is only~10–20-fold lower than that of native insulin (Hua et al., 1995Go), the insulin activity of IGF-1 swap is ~200-fold lower than that of native IGF-1. There is no immediately apparent reason why disulphide-swapping in IGF-1 should restrict its propensity for the conformational change needed for high-affinity insulin– receptor binding to a greater extent than does disulphide swapping in insulin. However, from the model it can be seen that the C-region has filled the space where the first A-region {alpha}-helix has uncoiled and this may be sterically hindering the interaction of Val44 with the second insulin receptor binding pocket. We propose to test this hypothesis by investigating the receptor binding affinities of IGF-1 molecules mutated at position 44, first to confirm the importance of this residue for insulin receptor binding and second to ascertain the effects of disulphide swapping on these mutants. It would also be interesting to generate the disulphide-swapped isomer of proinsulin to ascertain whether the presence of the C-peptide reduces its affinity for the insulin receptor to a greater extent than the reduction in affinity shown by insulin swap.


    Acknowledgments
 
Linda Larsø, Jürgen Stahl and Konstanze Thiemann provided excellent technical assistance. The competition data were fitted to a receptor dimer model by Dr Ronald Shymko. The CONTIN secondary structural analysis was carried out by Michael Jansen. Chandra Verma thanks the BBSRC for support.


    Notes
 
6 To whom correspondence should be addressed. E-mail, steve{at}soton.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received October 1, 1998; revised December 15, 1998; accepted January 5, 1999.