INTRODUCTION

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The binding of human TGF-¹ to the EGF receptor initiates a number of cell proliferation events including wound healing and embryogenesis (1, 2). Furthermore, this mitogenic protein of 50 residues is involved in the transformation of normal cells into malignant growths (3-5) and is also believed to promote angiogenesis (6). 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 (7, 8). Many attempts have also been made to synthesize reductant molecules that display a similar biological profile to the native TGF-, although for the most part these efforts have met with limited success (9-11). NMR structures of the free ligand have been determined by several groups (12-15), 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--EGFR extracellular domain complex (16, 17). The data from the latter experiments suggest that A and C loops and the C-terminal tail of TGF- contain residues (for the sequence of TGF- 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- does not play a role in the receptor-ligand interaction, the N-terminal deletion mutant, TGF- 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- 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- 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- where nonessential regions of the protein are deleted while retaining near native levels of receptor binding and activation.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the primary and loop structure of TGF-. The seven N-terminal residues removed in the deletion protein are indicated by shading as are the six cysteines that form the three disulfide loops. The disulfide bonds are represented as thick lines, and the loops formed by these are labeled as A, B, and C.

	EXPERIMENTAL PROCEDURES

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Synthesis and Purification of TGF- 8-50

TGF- 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 (10). 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.

NMR Sample Preparation

To a lyophilized sample of TGF- was added 460 µl of buffer containing 50 mM potassium phosphate, 10 mM potassium 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.

NMR Spectroscopy

One- and two-dimensional ¹H NMR spectra for TGF- 8-50 were collected on a Varian Unity spectrometer operating at 599.9 MHz. TOCSY (18) and NOESY (19) 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 (20). For TOCSY and NOESY experiments, 64 transients were acquired for each of 256 increments using the hypercomplex method of States et al. (21), 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 (22) 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- (23).

View this table: [in this window] [in a new window]   Table I 1H NMR assignments TGF- 8-50 at pH 6.5, 298 K

Structure Calculation of TGF- 8-50

For TGF- 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 code 1yug; Ref 15.) calculated using the programs NMRPipe (24) and PIPP (25) and a cut-off distance of 12 Å. The suite of programs CAMRA (26) 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 (27, 28) 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. (29) 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- 8-50 was examined using the program PROCHECK (30), which checks the distribution of backbone dihedral angles in the structures.

Binding and Activity of TGF- 8-50

Cell Culture-- 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 (31). The cell line was maintained in the Keratinocyte-SF (KSF) medium without supplements and incubated at 37 °C in an atmosphere containing 5% CO₂.

Proliferation Assay-- 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- or TGF- 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.

EGF Competition Assay-- 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-/TGF- 8-50 with 0.05 nM ¹²⁵I 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  counter. The nonspecific binding was determined in the presence of 1 µM unlabeled TGF- or TGF- 8-50. Data were analyzed using the method of Scatchard.

	RESULTS

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Analysis of Chemical Shift Variations between Truncated and Native TGF--- The deletion of the N-terminal tail of TGF- 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- 8-50 is present after synthesis and oxidation, the chemical shifts of the -protons were compared with those of the native protein acquired under similar conditions, i.e. pH 6.5 and 30 °C. The 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-. 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 -protons, those that exhibited the most significant changes were Gly¹⁹ and Thr²⁰ in addition to the -resonances of all the residues between Val²⁵ and Lys²⁹. For the hinge region of TGF- 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), 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 -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.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Plot of chemical shift variations () in the -protons between the truncated TGF- and the native protein for each residue in the molecule. Those residues with a value of greater than 0.1 ppm are denoted by white circles.

View larger version (69K): [in this window] [in a new window]   Fig. 3.   Aliphatic region of a NOESY spectrum of TGF- 8-50 acquired at 600 MHz and 25 °C. The NOE labeled is between the  protons of cysteines 8 and 21 and thus is indicative of correct formation of the disulfide bridges.

Receptor Binding and Activation by TGF- 8-50-- Investigation of the biological potency of TGF- 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- 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- 8-50 to displace iodinated EGF from HBE cells than it does for native TGF-, 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).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Plot of data from the competition assay of TGF- and TGF- 8-50. The graph illustrates the percentage of 125I-EGF displaced from the EGF receptor on Human BE cells as a function of ligand concentration.

NMR Structure Determination of TGF- 8-50-- As evidenced from the variation in the -H resonances between the N-terminally deleted and native TGF-s, the conformation of the truncated TGF- did not appear to be identical to that of previously determined structures of TGF-. With the objective of determining more precisely the structural differences that occur in TGF- 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 II and 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 (30) 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.

View this table: [in this window] [in a new window]   Table II Statistics for ensemble of NMR derived structures of TGF- 8-50

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Stereoview of the 35 best fit structures superimposed to the ensemble average structure calculated for the truncated TGF- using X-PLOR and NOE derived distance restraints. The more flexible regions of the protein are colored in gray.

	DISCUSSION

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Biological Potency of TGF- 8-50-- TGF- 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- 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- 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. (10), who previously synthesized TGF- 8-50 and reported binding and mitogenic activity of 3%. Our data imply that TGF- 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.

Solution Structure of TGF- 8-50-- The NMR-derived solution structure of TGF- 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 (12-15). The overall topology of the truncated protein superimposes favorably to that of the intact TGF- 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-s. The secondary structure elements present in TGF- remain in the structural ensemble of the N-terminally truncated molecule since the B loop -sheet comprised of residues 19-24 and 29-34 and the C-terminal reverse double hairpin loop between residues 38 and 46 were observed. The partial helix present in some of the TGF- conformations is no longer present in TGF- 8-50 because the A loop shows considerably flexibility. Some NOEs diagnostic of an -helix are observed for the truncated protein; however, these are insufficient to restrain the A loop into a helical conformation. The conformational differences of the A loop as observed in the structural ensembles are supported by the -H resonances, which show considerable variation when compared with the intact protein, and indeed as previously mentioned, the most significant changes in the resonance frequency of the -H of the N-terminally deleted molecule occur in this region.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Richardson diagrams (34, 35) of the average NMR structures calculated for wild type TGF- and TGF- 8-50. The center figure shows the superposition of the two ensemble average structures.

Implications of TGF- 8-50 Solution Structure for Functional Differences-- With the solution structure of TGF- 8-50 available, the biological rationale for decreased activity of the truncated protein as indicated by the reduction in receptor binding affinity and mitogenic activity can be envisaged. The previous studies from this laboratory implementing relaxation and NOE analysis techniques to elucidate the residues and regions of TGF- involved in complexation with EGFR (16, 17) were used to design the N-terminally deleted protein. The results obtained implicated residues from the A and C loops and C tail as forming the predominant receptor binding interface. Fig. 7 illustrates the average ensemble structures of TGF- and TGF- 8-50 shown as molecular Connolly surface representations and color coded according to the percentage of NOEs absent in the receptor-bound wild type TGF-, i.e. those present in the free ligand but not in the bound protein.

View larger version (101K): [in this window] [in a new window]   Fig. 7.   Grasp (36) molecular surface representation of the native and truncated TGF- average structures illustrating the residues color coded according to the percentage of NOEs, which disappear in the receptor-bound form of the native ligand. The residues are colored according to the following percentages of absent NOEs in the bound TGF-: 0%, blue; 0-20%, purple; 20-30%, white; 30-40%, green; and 40-60%, red.

Toward Minimization of TGF- through Truncation and Reoptimization-- Through the knowledge of the structural requirements of TGF- for receptor complexation as delineated by the relaxation rate and NOE analysis data, this study has demonstrated that the proposal of an active truncated protein is possible when enough is known about the receptor binding site on the ligand. The synthesis of a TGF- fragment with a respectable level of activity serves as a platform for further minimization and reoptimization of this growth factor for the purposes of the development of agonists and antagonists as potential therapeutics. Work currently in progress in this laboratory has the aim of engineering the truncated TGF- through phage display to increase the affinity to or beyond native levels (32). This can then be followed by further minimization of the ligand in a manner analogous to the studies of Li et al. (33), who have met with considerable success in the minimization of the polypeptide hormone atrial natriuretic peptide. In the instance of TGF-, however, the process is greatly aided by the availability of NMR data for elucidating the interactions of the minimized proteins with the receptor and thus using a structure-based iterative procedure for the identification of lead compounds demonstrating potential as therapeutics for diseases involving the overexpression or underexpression of TGF-.