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
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Abstract |
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Keywords: insulin/insulin-like growth factor-1/molecular dynamics/receptor binding/structurefunction relationships
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Introduction |
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The IGF-1 and insulin receptors are membrane glycoproteins composed of two extracellular -subunits and two transmembrane ß-subunits linked by disulphide bonds to give an (
ß)2 dimer (Massagué and Czech, 1982
; Ullrich et al., 1986
). 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., 1973
). 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., 1994
). 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 structurefunction relationships of insulin and IGF-1.
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Materials and methods |
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Cloning, expression and purification of human IGF-1 from S.cerevisiae has been described (Gill et al., 1996). 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 bufferacetonitrile 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 acetonitrilewaterpropan-2-olTFA gradient (Miller et al., 1993). The amino acid composition of each IGF-1 isomer was verified by amino acid analysis as described previously (Gill et al., 1996
).
SDSPAGE
SDSPAGE 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., 1992; Gill et al., 1996
). The binding data (percentage bound, duplicate determination) was fitted to a model assuming one high-affinity bound ligand per receptor dimer (Christoffersen et al., 1994
) from which the affinity (KD) was calculated.
Doseresponse 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., 1994). 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., 1996).
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., 1996). For comparison purposes, protein concentrations of the native and disulphide-swapped folds were normalized for tyrosine content (Balestrieri et al., 1978
). The far-UV CD spectra were analysed with respect to secondary structural composition using the CONTIN program (Provencher and Glöckner, 1981
).
Computer modelling and molecular dynamics of disulphide-swapped wild-type IGF-1
The wild-type IGF-1 model (Blundell et al., 1983) was used as the starting structure. The structure was modelled using the CHARMM empirical energy function (Brooks et al., 1983
) with the TIP3P model (Jorgensen et al., 1983
) for water and the parameter set 19 (Neria et al., 1996
) 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, 1993
; Ma and Karplus, 1997
) with large reductions in computational time. Positions of hydrogen atoms attached to the polar atoms were built using the HBUILD (Brünger and Karplus, 1988
) 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., 1983
). 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., 1984
). SHAKE (Ryckaert et al., 1977
; van Gunsteren and Karplus, 1982
) 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 NewtonRaphson minimizations until the gradient of the potential was
102 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.
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Results |
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Discussion |
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The far-UV CD spectrum of native IGF-1 is similar to that of insulin (Wood et al., 1975) 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
-helical content compared with native wild-type IGF-1. The analysis of the
-helical content of native IGF-1 (30%) agrees with that expected from the structure of IGF-1 in which residues 818, 4347 and 5458 are in a helical conformation (31.5%). The
-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., 1980). 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 (A20B19) and two left-handed disulphides (A7B7, A6A11) 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., 1993). 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., 1995
). Comparison of insulin swap with native insulin (Figure 9a
) reveals uncoiling of the first A-chain
-helix and a rearrangement in the orientation of the A and B chains by ~30° (Figure 9b
). Recent NMR studies of IGF-1 swap indicate that its structure is similar to that of insulin swap (Kobayashi, 1995
)
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 10a) 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
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., 1996
; Kazmirski and Dagget., 1998
). 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 10b
).
Just how valid is the model of IGF-1 swap and what does it tell us about its structurefunction relationships? The uncoiling of the first five residues of the first A-region -helix results in a total
-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 9b
); the side chain packing of Ile43 (A2 Ile) and Tyr60 (A19 Tyr) is maintained despite the loss of
-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 -helical conformation to orientate A3 Val for interaction with a second receptor pocket (Nakagawa and Tager, 1992
). 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, 1994
). 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., 1991
). 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, 1990
).
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~1020-fold lower than that of native insulin (Hua et al., 1995), 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
-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.
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Acknowledgments |
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Notes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anfinsen,C.B. (1973) Science, 181, 223230.[ISI][Medline]
Axelsson,K., Johansson,S., Eketorp,G., Zazzi,H., Hemmendorf,B. and Gellerfors,P. (1992) Eur. J. Biochem., 206, 987994.[Abstract]
Balestrieri,C., Colonna,G., Giovane,A., Irace,G. and Servillo,L. (1978) Eur. J. Biochem., 90, 433442.[Abstract]
Baker,E.N. et al. (1988) Philos. Trans. R. Soc. London, B319, 369456.[ISI]
Berendsen,H.J.C., Potsma,P.M., van Gunsteren,W.F., DiNola,A. and Haak,J.R. (1984) J. Chem. Phys., 81, 36843690.[ISI]
Blundell,T.L., Berdarkar,S., Rinderknecht,E.and Humbel RE. (1978) Proc. Natl Acad. Sci. USA, 75, 180184.[Abstract]
Blundell,T.L., Berdarkar,S. and Humbel,R.E. (1983) Fed. Proc., 42, 25922597.[ISI][Medline]
Brooks,B., Bruccoleri,R.E., Olafson,B.D., States,D.J., Swaminathan,S. and Karplus,M. (1983) J. Comput. Chem., 4, 187217.[ISI]
Bruccoleri,R.E. and.Karplus,M. (1986) J. Comput. Chem., 87, 165175.
Brünger,A.T. and Karplus,M. (1988) Proteins: Struct. Funct. Genet., 4, 148156.[ISI][Medline]
Cascieri,M.A. and Bayne,M.L. (1990) In Le Roth,D. and Raizada,M.K. (eds), Molecular and Cellular Biology of IGFs and their Receptors. Plenum Press, London, pp. 2330.
Christoffersen,C.T., Bornfeldt,K.E., Rotella,C.M., Gonzales,N., Vissing,H., Shymko,R.M., ten Hoeve,J., Groffen,J., Heisterkamp,H. and De Meyts,P. (1994) Endocrinology, 135, 472475.[Abstract]
Cooke,R.M., Harvey,T.S. and Campbell,I.D. (1991) Biochemistry, 30, 54845491.[ISI][Medline]
De Meyts,P. (1994) Diabetologia, 37 (Suppl. 2), S135S148.[ISI][Medline]
De Meyts,P., Roth,J., Neville,D.M., Gavin,J.R. and Lesniak,M.A. (1973) Biochem. Biophys. Res. Commun., 55, 154161.[ISI][Medline]
Elamrani,S., Berry,M.B., Phillips,G.N. and McCammon,J.A. (1996) Proteins: Struct. Funct. Genet., 25, 7988.[ISI][Medline]
Elliot,S. et al. (1990) J. Protein Chem., 9, 95104.[ISI][Medline]
Froesch,E.R., Schmid,C., Schwander,J. and Zapf,J. (1985) Annu. Rev. Physiol., 47, 443467.[ISI][Medline]
Forsberg,G., Palm,G., Ekebacke,A., Josephson,S. and Hartmanis,M. (1990) Biochem. J., 271, 357363.[ISI][Medline]
Gill,R. et al. (1996) Protein Engng, 9, 10111019.[Abstract]
Gellerfors,P., Axelsson,K., Helander,A., Johansson,S., Lennart,K., Lindquist,S., Parlu,B., Skottner,A. and Fryklund,L. (1989) J. Biol. Chem., 264, 1144411449.
Hua,Q.-X., Gozani,S.N., Chance,R.E., Hoffman,J.A., Frank,B.H. and Weiss,M.A. (1995) Nature Struct. Biol., 2, 129138.[ISI][Medline]
Iwai,M., Kobayashi,M., Tamura,K., Ishi,Y., Yamada,H. and Niwa,M. (1989) J. Biochem., 106, 949957.[Abstract]
Jorgensen,W.L., Chandrasekhar,J., Medura,J.D., Impey,R.W. and Klein,M.L. (1983) J. Chem. Phys., 79, 926935.[ISI]
Kasuya,J., Paz,I.B., Maddux,B.A., Goldfine,I.D., Hefta,S.A. and Fujita-Yamaguchi,Y. (1993) Biochemistry, 32, 1353113536.[ISI][Medline]
Kazmirski,S.L. and Dagget,V. (1998) J. Mol. Biol., 277, 487506.[ISI][Medline]
Kjeldsen,T. Andersen,A.S., Wiberg,F.C., Rasmussen,J.S., Schäffer,L., Balschmidt,P., Møller,K.B. and Møller,N.P.H. (1991) Proc. Natl Acad. Sci. USA, 88, 44044408.[Abstract]
Kobayashi,Y. (1995) Cited in Hua et al. (1995).
Kraulis, P.J. (1991) J. Appl. Crystallogr., 24, 946950.[ISI]
Linderberg,J. and Michl,J. (1970) J. Am. Chem. Soc., 92, 26192625.[ISI]
Ma,J. and Karplus,M. (1997) J. Mol. Biol., 274, 114131.[ISI][Medline]
Massagué,J. and Czech,M.P. (1982) J. Biol. Chem., 257, 50385045.
Meng,H., Burleigh,B.D. and Kelly,G.M. (1988) J. Chromatogr., 443, 183192.[Medline]
Miller,J.A., Narhi,L.O., Hua,Q.-X., Rosenfeld,R., Arakawa,T., Rohde,M., Prestreleski,S., Lauren,S., Stoney,K.S., Tsai,L. and Weiss,M.A. (1993) Biochemistry, 32, 52035213.[ISI][Medline]
Nakagawa,S.H. and Tager,H.S. (1992) Biochemistry, 31, 32043214.[ISI][Medline]
Neria,E., Fischer,S. and Karplus,M. (1996) J. Chem. Phys., 105, 19021921.[ISI]
Provencher,S.W. and Glöckner,J. (1981) Biochemistry, 30, 3337.
Ryckaert,J.-P., Ciccotti,G. and Berendesen,H.J.C. (1977) J. Comput. Phys., 23, 327341.[ISI]
Sato,A., Nishimura,S., Ohkubo,T., Kyogoku,Y., Koyama,S., Kobayashi,M., Yasuda,T. and Kobayashi,Y. (1993) Int. J. Pept. Protein Res., 41, 433440.[ISI][Medline]
Steinbach, P.J. and Brooks, B.R. (1993) Proc. Natl Acad. Sci. USA, 90, 91359139.[Abstract]
Tamura,K., Kobayashi,M., Koyama,S., Yamada,H., Ishi,Y., Niwa,M. and Iwai,M., (1988) 8th International Congress of Endocrinology, Kyoto, Japan, Abstr. 2318040.
Ullrich,A. et al. (1986) EMBO J., 5, 25032512.[Abstract]
van Gunsteren,W.F. and Karplus,M. (1982) Macromolecules, 15, 15281544.[ISI]
Wollmer,A. et al. (1980) In Brandenburg,D. and Wollmer,A. (eds), Insulin, Chemistry, Structure and Function of Insulin and Related Hormones. Walter de Gruyter, Berlin, pp. 2735.
Wood,S.P., Blundell,T.L., Wollmer,A., Lazarus,N.R. and Neville,R.W.J. (1975) Eur. J. Biochem., 55, 531542.[Abstract]
Received October 1, 1998; revised December 15, 1998; accepted January 5, 1999.