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W2087759737 abstract "The investigation of a N-terminally truncated human transforming growth factor-α (TGF-α; residues 8–50) has been completed to determine the contribution of the N terminus to receptor binding and activation. The deletion protein was proposed and designed through study of NMR relaxation and nuclear Overhauser enhancement data obtained from the TGF-α-epidermal growth factor (EGF) receptor complex, which indicated that the residues N-terminal to the A loop remain flexible in receptor-bound TGF-α and thus suggested their lack of involvement in receptor binding (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283–15292; McInnes, C., Hoyt, D. W., Harkins, R. N., Pagila, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1996) J. Biol. Chem. 271, 32204–32211). TGF-α 8–50 was shown to have approximately 10-fold lower affinity for the receptor than the native molecule in an assay quantifying the ability to compete with EGF for binding and to have a similar reduction in activity as indicated by a cell proliferation assay. NMR solution structural calculations on this molecule demonstrate correct formation of the three disulfide bonds of TGF-α 8–50 and have established the presence of native secondary structure in the B and C loops of the protein. However, some perturbation of the global fold with respect to the orientation of the subdomains was observed. These results suggest that although the N-terminal residues do not contribute directly to binding, they make a significant contribution in defining the conformation of the growth factor, which is required for complete binding and activity and is therefore significant in terms of producing native folding of TGF-α. They also show that information obtained from the receptor-bound ligand can be used to guide the design and minimization of TGF-α analogues. The implications of the study of TGF-α 8–50 for the design and synthesis of reductants of this growth factor are therefore discussed. The investigation of a N-terminally truncated human transforming growth factor-α (TGF-α; residues 8–50) has been completed to determine the contribution of the N terminus to receptor binding and activation. The deletion protein was proposed and designed through study of NMR relaxation and nuclear Overhauser enhancement data obtained from the TGF-α-epidermal growth factor (EGF) receptor complex, which indicated that the residues N-terminal to the A loop remain flexible in receptor-bound TGF-α and thus suggested their lack of involvement in receptor binding (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283–15292; McInnes, C., Hoyt, D. W., Harkins, R. N., Pagila, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1996) J. Biol. Chem. 271, 32204–32211). TGF-α 8–50 was shown to have approximately 10-fold lower affinity for the receptor than the native molecule in an assay quantifying the ability to compete with EGF for binding and to have a similar reduction in activity as indicated by a cell proliferation assay. NMR solution structural calculations on this molecule demonstrate correct formation of the three disulfide bonds of TGF-α 8–50 and have established the presence of native secondary structure in the B and C loops of the protein. However, some perturbation of the global fold with respect to the orientation of the subdomains was observed. These results suggest that although the N-terminal residues do not contribute directly to binding, they make a significant contribution in defining the conformation of the growth factor, which is required for complete binding and activity and is therefore significant in terms of producing native folding of TGF-α. They also show that information obtained from the receptor-bound ligand can be used to guide the design and minimization of TGF-α analogues. The implications of the study of TGF-α 8–50 for the design and synthesis of reductants of this growth factor are therefore discussed. transforming growth factor-α epidermal growth factor epidermal growth factor receptor nuclear Overhauser enhancement nuclear Overhauser enhancement spectroscopy total correlation spectroscopy high pressure liquid chromatography root mean square. The binding of human TGF-α1 to the EGF receptor initiates a number of cell proliferation events including wound healing and embryogenesis (1Bennet N.T. Schultz G.S. Am. J. Surg. 1993; 165: 728-737Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 2Bennet N.T. Schultz G.S. Am. J. Surg. 1993; 166: 75-81Google Scholar). Furthermore, this mitogenic protein of 50 residues is involved in the transformation of normal cells into malignant growths (3Prigent S.A. Lemoine N.R. Prog. Growth. Factor. Res. 1992; 4: 1-24Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 4Salomon D.S. Brandt R. Ciardiello F. Normanno N. Crit. Rev. Oncol. Hematol. 1995; 19: 183-232Crossref PubMed Scopus (2411) Google Scholar, 5Rusch V. Mendelsohn J. Dmitrovsky E. Cytokine Growth Factor. Rev. 1996; 7: 133-141Crossref PubMed Scopus (99) Google Scholar) and is also believed to promote angiogenesis (6Okamura K. Morimoto A. Hamanaka R. Ono M. Kohno K. Uchida Y. Kuwano M. Biochem. Biophys. Res. Commun. 1992; 186: 1471-1479Crossref PubMed Scopus (79) Google Scholar). It is thus apparent that this polypeptide is a significant target from a pharmaceutical and drug design perspective, and to this end, considerable effort has been made to elucidate the essential structural features of this tricyclic growth factor required for binding and function (7Groenen L.C. Nice E.C. Burgess A.W. Growth Factors. 1994; 11: 235-257Crossref PubMed Scopus (215) Google Scholar, 8Campion S.R. Niyogi S.K. Prog. Nucleic Acids Res. 1994; 49: 353-383Crossref PubMed Scopus (28) Google Scholar). Many attempts have also been made to synthesize reductant molecules that display a similar biological profile to the native TGF-α, although for the most part these efforts have met with limited success (9Defeo-Jones D. Tai J.Y. Wegrzyn R.J. Vuocolo G.A. Baker A.E. Payne L.S. Garsky V.M. Oliff A. Rieman M.W. Mol. Cell. Biol. 1988; 8: 2999-3007Crossref PubMed Scopus (47) Google Scholar, 10Tam J.P. Lin Y.-Z. Liu W. Wang D.-X. Ke X.-H. Zhang J.-W. Int. J. Pept. Protein Res. 1991; 38: 204-211Crossref PubMed Scopus (16) Google Scholar, 11Heath W.F. Merrifield R.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6367-6371Crossref PubMed Scopus (72) Google Scholar). NMR structures of the free ligand have been determined by several groups (12Kohda D. Shimada I Miyake T. Fuwa T. Inagaki F. Biochemistry. 1989; 28: 953-958Crossref PubMed Scopus (42) Google Scholar, 13Kline T.P. Brown F.K. Brown S.C. Jeffs P.W. Kopple K.D. Mueller L. Biochemistry. 1990; 29: 7805-7813Crossref PubMed Scopus (55) Google Scholar, 14Harvey T.S. Wilkinson A.J. Tappin M.J. Cooke R.M. Campbell I.D. Eur. J. Biochem. 1991; 198: 555-562Crossref PubMed Scopus (54) Google Scholar, 15Moy F.J. Li Y-C. Rauenbuehler P. Winkler M.E. Scheraga H.A. Montelione G.T. Biochemistry. 1993; 32: 7334-7353Crossref PubMed Scopus (60) Google Scholar), and recently studies have been published from this laboratory on the use of NMR relaxation and NOE measurements in determining the essential components of the TGF-α-EGFR extracellular domain complex (16Hoyt D.W. Harkins R.N. Debanne M.T. O'Connor-McCourt M. Sykes B.D. Biochemistry. 1994; 33: 15283-15292Crossref PubMed Scopus (28) Google Scholar, 17McInnes C. Hoyt D.W. Harkins R.N. Pagila R.N. Debanne M.T. O'Connor-McCourt M. Sykes B.D. J. Biol. Chem. 1996; 271: 32204-32211Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The data from the latter experiments suggest that A and C loops and the C-terminal tail of TGF-α contain residues (for the sequence of TGF-α and location of disulfides forming the three loops, see Fig. 1) that form the major binding interface with the receptor and that the N-terminal amino acids outside the A loop remain flexible in the receptor-bound species. Since the consensus from the relaxation and NOE data was that the N terminus of TGF-α does not play a role in the receptor-ligand interaction, the N-terminal deletion mutant, TGF-α 8–50 was synthesized and characterized in terms of NMR structure and biological activity. These experiments were performed to clarify the requirements of the growth factor structure necessary for receptor binding and activation and to determine the contribution of the N-terminal tail to the formation of the global fold of the native molecule. Thus the rationale is to assess any functional changes of the truncated protein with respect to native TGF-α in terms of structural variation and concurrently in doing this to ascertain if the N-terminal tail residues are required for establishing the native conformation of the protein. The truncated TGF-α was also studied to demonstrate that structural information obtained from NMR experiments on the receptor-bound ligand can be used toward the design of a “minimized” TGF-α where nonessential regions of the protein are deleted while retaining near native levels of receptor binding and activation. TGF-α 8–50 was synthesized using standard solid phase synthesis techniques on a Applied Biosystems model 430A peptide synthesizer (Foster City, CA) utilizing t-butoxycarbonyl methodology. The N terminus was acetylated using acetic anhydride, and the peptide was liberated from the resin using a mixture of hydrogen fluoride, anisole and 1,2-ethanedithiol. The crude material was extracted with glacial acetic acid, lyophilized, and then dissolved in 10% acetic acid. The peptide was then purified using reversed phase HPLC (Beckmann System Gold, Fullerton, CA) using a Synchropak RP-4 (250 × 21.2 mm inner diameter) column (Synchrom, Lafayette, IN) at a flow rate of 5 ml/min and subsequently oxidized in air using a solution containing 1.0 m urea, 0.1 m Tris, 1.5 nm oxidized and 0.75 nm reduced glutathione, pH 8.0 (10Tam J.P. Lin Y.-Z. Liu W. Wang D.-X. Ke X.-H. Zhang J.-W. Int. J. Pept. Protein Res. 1991; 38: 204-211Crossref PubMed Scopus (16) Google Scholar). A final purification step was used to remove oxidation byproducts, and the final purity was confirmed through reverse phase analytical HPLC. Electrospray mass spectrometry (VG Biotech, Cheshire, UK) verified the presence of a species of the desired molecular weight. To a lyophilized sample of TGF-α was added 460 μl of buffer containing 50 mm potassium phosphate, 10 mmpotassium chloride, 1 mm ethylene diamine tetraacetic acid, 0.5 mm sodium azide, 0.15 mm sodium 2,2-dimethyl-2-silapentane-5-sulfonate (internal standard), and 99.9% D2O or 90%/10% (v/v) H2O/D2O. The solution was adjusted to pH 6.5 by the addition of small aliquots of 0.5 n NaOD or 0.5 n HCl bringing the final volume to 500 μl. One- and two-dimensional 1H NMR spectra for TGF-α 8–50 were collected on a Varian Unity spectrometer operating at 599.9 MHz. TOCSY (18Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) and NOESY (19Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4809) Google Scholar) experiments were acquired at 298 K and referenced relative to an internal sodium 2,2-dimethyl-2-silapentane-5-sulfonate standard. Pulsed field gradients were implemented in the watergate pulse sequence to suppress the water resonance (20Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3487) Google Scholar). For TOCSY and NOESY experiments, 64 transients were acquired for each of 256 increments using the hypercomplex method of States et al. (21States D.J. Haberkorn R.A. Rueben D.J. J. Magn. Reson. 1982; 48: 286-292Google Scholar), and a total of 4096 data points were collected over a spectral width of 8000 Hz for H2O spectra and 6000 Hz for D2O spectra. The SCUBA-NOESY (22Brown S.C. Weber P.L. Mueller K. J. Magn. Reson. 1988; 77: 166-169Google Scholar) experiment was utilized for acquisition of D2O NOE data. Processing of each two-dimensional FID was accomplished using a shifted sinebell and zero filling to 4096 points in both F1 and F2. A complete resonance assignment was obtained from the TOCSY and NOESY data with the exception of the fast exchanging His12 amide proton and is shown in Table I. The assignment was assisted through the use of chemical shift information previously published for native TGF-α (23Tappin M.J. Cooke R.M. Fitton J.E. Campbell I.D. Eur. J. Biochem. 1989; 179: 629-637Crossref PubMed Scopus (27) Google Scholar).Table I1H NMR assignments TGF-α 8–50 at pH 6.5, 298 KResidueChemical shiftHNHαHβOther protonsCys88.504.403.06, 2.97CH3CO2 1.83Pro94.442.21, 1.94Hγ 1.72, 1.72; Hδ3.36, 3.24Asp108.484.442.69, 2.69Ser118.194.253.82, 3.82His124.673.34, 2.98H2 8.00 H4 6.85Thr137.794.214.15Hγ 1.19Gln148.404.211.99, 1.81Hγ 2.13, 2.13; HεN 7.36, 6.75Phe157.984.203.06, 2.85H2,6 7.17 H3,5 7.18Cys168.214.332.46, 2.04Phe178.334.222.69, 2.69H2,6 7.10; H3,5 7.19His186.874.452.72, 2.25H2 8.34, H4 6.86Gly197.353.90, 3.51Thr208.414.513.95Hγ 1.27Cys218.995.143.25, 3.14Arg229.434.611.72, 1.54HγHδPhe238.934.593.01, 2.86H2,6 6.86; H3,5 7.16; H4 7.24Leu248.284.431.77, 1.46Hγ 1.55; H3δ 0.76, 0.72Val258.153.682.08H3γ1.12, 1.00Gln268.744.102.08, 2.08Hγ 2.40, 2.40; HεN.7.63, 6.86Glu277.704.211.73Hγ 2.19, 2.06Asp287.984.283.07, 2.38Lys297.054.721.74, 1.58Hγ 1.24, 1.24; Hδ Hε 2.95, 2.95Pro304.711.76, 1.66Hγ 1.45, 1.45; Hδ 3.54, 3.43Ala318.774.731.37Cys328.785.202.77, 2.62Val339.044.232.05H3γ0.95, 0.92Cys349.135.063.44, 2.74His358.724.873.40, 2.88H2 7.78; H4 7.01Ser368.664.273.92, 3.92Gly378.824.15, 3.56Tyr388.025.282.92, 2.77H2,6 6.67; H3,5 6.40Val399.264.852.28H3γ0.86, 0.79Gly408.105.04, 3.90Ala419.244.181.56Arg428.834.712.26, 2.26Hγ 1.37, 0.91; HδCys438.093.962.92, 2.44Glu4410.054.251.62, 1.62Hγ 1.83, 1.37His458.555.133.08, 2.99H28.10; H4 7.03Ala468.734.171.15Asp478.124.422.48, 2.21Leu488.074.251.58, 1.58Hγ 1.57; H3δ 0.88, 0.82Leu498.194.311.64, 1.57Hγ 1.58; H3δ 0.87, 0.82Ala507.594.051.28 Open table in a new tab For TGF-α 8–50, all cross-peaks were volume integrated using the program VNMR (VNMR 5.1A, Varian Associates, Palo Alto, CA) of which a subset were assigned based on the chemical shift assignments and possible NOE distances in the native protein (Protein Data Bank code1yug; Ref 15Moy F.J. Li Y-C. Rauenbuehler P. Winkler M.E. Scheraga H.A. Montelione G.T. Biochemistry. 1993; 32: 7334-7353Crossref PubMed Scopus (60) Google Scholar.) calculated using the programs NMRPipe (24Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11280) Google Scholar) and PIPP (25Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (799) Google Scholar) and a cut-off distance of 12 Å. The suite of programs CAMRA (26Grönwald, W., Willard, L., Jellard, T., Boyko, R. F., Rajarathnam, K, Wishart, D. S., Sönnichsen, F. D., and Sykes, B. D. (1998) J. Biol. NMR, in pressGoogle Scholar) was implemented to convert the volume integral information to distance restraints. NOE cross-peak intensities were classified as strong (upper boundary, 2.3 Å), medium (3.0 Å), weak (4.0 Å), and very weak (5.0 Å), and the appropriate pseudoatom correction was added. The molecular dynamics/simulated annealing protocol in X-PLOR version 3.8 (27Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (767) Google Scholar, 28Brünger A.T. X-Plor, version 3.1. Yale University Press, New Haven, CT1993Google Scholar) was utilized to calculate an initial set of structures derived only from the unambiguous distance restraints and native disulfides assumed in the starting structure. A program developed in house was then applied in conjunction with X-PLOR to modify NOE distances causing restraint violations and also to make use of those that have ambiguous assignments to generate a more refined structure. This program uses an approach similar to that of Nilges et al. (29Nilges M. Macias M.J. O' Donoghue S.I. Oschkinat H. J. Mol. Biol. 1997; 269: 408-422Crossref PubMed Scopus (387) Google Scholar) in that it allows the use of NOE distances that are ambiguous, because in most cases only one restraint will be possible based on distances from the ensemble of structures already calculated using unambiguous restraints. The program therefore removes restraints that are violated by greater than a user defined cut-off distance (0.5 Å) because the nonpossible assignments for the ambiguous restraints will give distance violations greater than the cut-off. The restraints that satisfy the distances in the initial structures will be retained. Because it is possible that a correct restraint can be violated in initial but not final structures, NOEs that are removed can be returned to the restraint file in later runs for reevaluation by the program. As the structures are refined in successive runs, these restraints should now be satisfied and are incorporated in the final data set. This procedure as a whole allows the restraints that satisfy the distances in the successive structural ensembles to be retained and thus facilitates the incorporation of a greater number of restraints in the X-PLOR calculation. For the distance restraints that are consistently violated by less than the specified cut-off in the calculated structures, the program automatically modifies the upper bound of distance restraints by the average value of the distance violation for a user specified number of structures in which the violation occurs. The modified restraint file is then used in the next round of X-PLOR calculations in an iterative procedure until an structural ensemble is obtained of the desired quality. The reproducibility and accuracy of the ensemble generated for TGF-α 8–50 was examined using the program PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-290Crossref Google Scholar), which checks the distribution of backbone dihedral angles in the structures. The HBE4-E6E7 cell line was used and was established by transfection of primary human bronchial epithelial cells with a plasmid construct expressing the human papillomavirus type 16 E6 and E7 genes (31Viallet J. Liu C. Emond J. Tsao M.S. Exp. Cell Res. 1994; 212: 36-41Crossref PubMed Scopus (51) Google Scholar). The cell line was maintained in the Keratinocyte-SF (KSF) medium without supplements and incubated at 37 °C in an atmosphere containing 5% CO2. Growth curves were established by measuring the incorporation of [3H]methylthymidine into the acid insoluble fraction of cellular extracts. Briefly, approximately 2 × 104 cells were plated in 1 ml KSF into each well of 12-well tissue culture plates. After 1 day, the medium was changed either with fresh KSF (control) or medium containing TGF-α or TGF-α 8–50. Cells were allowed to grow for 3 days with [3H]methylthymidine (1 μCi/ml, Amersham Canada) being added during the last 20–24 h. After sequential and multiple washings in cold phosphate-buffered saline, 10% trichloroacetic acid, and 95% ethanol, the plates were allowed to dry, the cells were solubilized in 2% SDS, and the radioactivity was quantitated by liquid scintillation chromatography. The experimental points were determined in triplicate. Cells (2 × 103) were plated in 24-well plates in KSF and grown to confluence. 24 h before performing the experiment, the medium was changed with KSF. Cells were washed three times with binding buffer containing Dulbecco's modified Eagle's medium, 25 mm Hepes, 5 mm MgCl2, 100 μg/ml bacitracin, 0.1% bovine serum albumin, pH 7.4, and incubated in 0.5 ml of binding buffer containing various concentrations of TGF-α/TGF-α 8–50 with 0.05 nm125I EGF at 0 C for 2 h. After incubation, the cells were washed twice with ice-cold binding buffer and three times with ice-cold phosphate-buffered saline. Cells were solubilized with 1 n NaOH and counted in a γ counter. The nonspecific binding was determined in the presence of 1 μm unlabeled TGF-α or TGF-α 8–50. Data were analyzed using the method of Scatchard. The deletion of the N-terminal tail of TGF-α raises the question of whether or not native disulfide bonds are present in the truncated form of the protein and if it is able to assume a similar global fold to the intact protein. For the purpose of determining whether the correct pairing of the disulfides in TGF-α 8–50 is present after synthesis and oxidation, the chemical shifts of the α-protons were compared with those of the native protein acquired under similar conditions, i.e. pH 6.5 and 30 °C. The δΔ values of the comparison (chemical shift differences) are illustrated in Fig. 2, and it can be seen that there are no large deviations in these shifts. This suggests that the three disulfide links between Cys8 and Cys21, Cys16 and Cys32, and Cys34 and Cys43 are present as observed in the intact TGF-α. The presence of the native disulfide linkages was confirmed through observation of NOEs between cysteines 8 and 21 (Fig. 3) and 34 and 43 (not shown). Also from the absence of major deviations the assumption can be made that a structure cognate to the native molecule exists. However, despite the lack of large differences in the chemical shifts of the deletion mutant, significant changes of between 0.05 and 0.2 ppm are observed. These occur for the most part in the A and B loops and indicate that deletion of the residues N-terminal to the A loop effects changes in the secondary structure and perhaps in the overall fold of the molecule. In particular, the residues undergoing the largest variation are those of the A loop including Tyr13, Gln14, and Phe15, which have resonances differing from the native protein by 0.17, 0.13, and 0.13 ppm respectively. Of the B loop α-protons, those that exhibited the most significant changes were Gly19 and Thr20 in addition to the α-resonances of all the residues between Val25 and Lys29. For the hinge region of TGF-α 8–50 (residues close to Val33), which allows flexibility between the two subdomains (A and B loops comprise the N subdomain, whereas the C loop and tail form the C subdomain), δΔ values of between 0.04 and 0.09 ppm were observed for the residues between Cys32 and Ser36 and thus are indicative of a possible variation in the conformation of this region. An important observation of the shifts for the residues of the C loop is that with the exception of Gly40 and Glu44 there are no differences larger than 0.05 ppm, suggesting that the reverse double hairpin secondary structure is present in the truncated protein. The δΔ α-H value for Glu44 of 0.08 ppm is of special interest because this residue forms interactions critical to the stabilization of the orientation of the N- and C-terminal subdomains.Figure 3Aliphatic region of a NOESY spectrum of TGF-α 8–50 acquired at 600 MHz and 25 °C. The NOE labeled is between the β protons of cysteines 8 and 21 and thus is indicative of correct formation of the disulfide bridges.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Investigation of the biological potency of TGF-α 8–50 was performed to assess the contribution of the N-terminal tail to receptor binding and activation. This was accomplished through a competition assay of TGF-α 8–50 with EGF on HBE cells displaying the EGF receptor and by measuring the ability of the truncation analogue to effect cell proliferation. The results from the competition assay are shown in Fig. 4. From the plot it is apparent that it requires approximately 10-fold more TGF-α 8–50 to displace iodinated EGF from HBE cells than it does for native TGF-α, thus indicating that it has 10 times lower affinity for the EGF receptor. The quantitation of the receptor activation assay, however, illustrates that the reduction in the ability of the truncated protein to cell proliferation is of the same order as the corresponding decrease in receptor binding (data not shown). As evidenced from the variation in the α-H resonances between the N-terminally deleted and native TGF-αs, the conformation of the truncated TGF-α did not appear to be identical to that of previously determined structures of TGF-α. With the objective of determining more precisely the structural differences that occur in TGF-α 8–50, an ensemble of solution structures was calculated for the N-terminally deleted protein using NOE-derived distance restraints. 35 best fit structures were generated from 407 NOE distances using the program X-plor after nine rounds of calculation to minimize restraint violations and make the best use of ambiguous NOEs. The structure statistics for the ensemble of 35 structures are shown in Table IIand illustrate their quality in terms of high definition, in terms of fit to the experimental data, and in terms of the component and total energies. Analysis of the average of the 35 structures using the program PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-290Crossref Google Scholar) indicated that 92% of the residues excluding glycine, proline, and end residues are found in the allowed regions of phi-psi space. The residues found in non-allowed regions, which are Phe15 and Cys16, are located in the highly flexible portions of the truncated molecule.Table IIStatistics for ensemble of NMR derived structures of TGF-α 8–50XPLOR energies (kcal/mol) Total energy73.5 ± 0.326 Bond energy3.12 ± 0.018 Angle energy57.0 ± 0.167 Improper energy5.01 ± 0.029 van der Waals energy3.72 ± 0.057 NOE energy4.64 ± 0.058r.m.s. deviations from ideal geometry (Å) Bond0.00224 ± 0.00001 Angle0.580 ± 0.00116 Improper0.309 ± 0.00109r.m.s. deviations from experimental restraints (Å) Total NOE restraints (409)0.0147 ± 0.00010 Intra-residue (154)0.0116 ± 0.0062 Sequential (126)0.00730 ± 0.0036 Medium range (33)0.0181 ± 0.0043 Long range (94)0.0113 ± 0.0029r.m.s. deviations of cartesian coordinates (§) (35 structures vs. the average structure) Residues 15–47 (backbone)0.844 ± 0.119 Residues 15–47 (heavy)1.304 ± 0.144 Residues 15–47 (all)1.477 ± 0.135 Residues 19–24, 29–34, 38–46 (backbone)0.654 ± 0.095 Residues 19–24, 29–34, 38–46 (heavy)1.083 ± 0.131 Residues 19–24, 29–34, 38–46 (all)1.304 ± 0.126 Open table in a new tab Fig. 5 shows a superposition of the 35 best fit structures to the average structure and depicts the excellent reproducibility of the conformational ensemble. The values for the superposition of residues 15–47 for the 35 structures to the average structure are 0.844 Å for the backbone atom r.m.s. deviations and 1.304 for the heavy atom r.m.s. deviations, which are very favorable in their comparability with the ensemble recently generated for TGF-α using similar conditions for data acquisition (15Moy F.J. Li Y-C. Rauenbuehler P. Winkler M.E. Scheraga H.A. Montelione G.T. Biochemistry. 1993; 32: 7334-7353Crossref PubMed Scopus (60) Google Scholar). TGF-α 8–50 was proposed and designed based on the results of the relaxation and NOE analysis data of the ligand-receptor complex, which suggested that the N terminus does not contribute to the complexation process and that the truncated protein should retain the ability to associate with the EGF receptor and initiate cell proliferation. Our results indicate that although the binding and activation are diminished 10-fold, TGF-α 8–50 still retains a significant level of potency in terms of competition with EGF for the receptor and ability to effect cell proliferation. The plethora of studies on synthetic fragments of TGF-α have for the most part failed to exhibit biological activity close to the order of that observed for the native molecule. Our results differ slightly from those of Tam et al. (10Tam J.P. Lin Y.-Z. Liu W. Wang D.-X. Ke X.-H. Zhang J.-W. Int. J. Pept. Protein Res. 1991; 38: 204-211Crossref PubMed Scopus (16) Google Scholar), who previously synthesized TGF-α 8–50 and reported binding and mitogenic activity of 3%. Our data imply that TGF-α 8–50 is more potent; however, this variation may be the result of different methods used for assaying activity. In our case, the protein synthesized in this laboratory was fully characterized by NMR and thus the possibility of ambiguity in the structure and correct folding of the disulfides is not present, and therefore the biological data are reliable in terms of the integrity of the deletion protein. The NMR-derived solution structure of TGF-α 8–50 exhibits similar structural characteristics in comparison with the global fold of the native protein as has been determined by several groups to date (12Kohda D. Shimada I Miyake T. Fuwa T. Inagaki F. Biochemistry. 1989; 28: 953-958Crossref PubMed Scopus (42) Google Scholar, 13Kline T.P. Brown F.K. Brown S.C. Jeffs P.W. Kopple K.D. Mueller L. Biochemistry. 1990; 29: 7805-7813Crossref PubMed Scopus (55) Google Scholar, 14Harvey T.S. Wilkinson A.J. Tappin M.J. Cooke R.M. Campbell I.D. Eur. J. Biochem. 1991; 198: 555-562Crossref PubMed Scopus (54) Google Scholar, 15Moy F.J. Li Y-C. Rauenbuehler P. Winkler M.E. Scheraga H.A. Montelione G.T. Biochemistry. 1993; 32: 7334-7353Crossref PubMed Scopus (60) Google Scholar). The overall topology of the truncated protein superimposes favorably to that of the intact TGF-α as shown by backbone r.m.s. deviations of 2.78 Å for residues 15–47. The identity between the two structures is demonstrated in Fig. 6, which illustrates the Richardson diagrams of the native and N-terminally deleted TGF-αs. The secondary structure elements present in TGF-α remain in the structural ensemble of the N-terminally truncated molecule si" @default.
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