TGF-β2 uses the concave surface of its extended finger region to bind betaglycan's ZP domain via three residues specific to TGF-β and inhibin-α

Assignment and secondary structure of TGF-β2

TGF-βs, and most other GFs of the TGF-β family, have micromolar or lower solubilities from about pH 4.5 to about 10 because of the hydrophobic nature of their surfaces and their close to neutral charge over this range (36, 37). The poor solubility is known to be further aggravated by the presence of salts, such as NaCl (37). Thus, the previous NMR studies of TGF-β1 and TGF-β3 were carried out under acidic conditions in the absence of salts, 95% H₂O, 5% ²H₂O, pH 4.2, 45 °C for TGF-β1 and 87% H₂O, 5% ²H₂O, 8% [²H]dioxane, 3% [²H]methanol, pH 2.9, 45 °C for TGF-β3, where they were both highly soluble and structured (38, 39). To identify the ideal conditions for TGF-β2, we collected spectra of ¹⁵N TGF-β2 as the pH was varied from 2 to 5 and the temperature was varied from 25 to 50 °C. We found the conditions that led to the most dispersed ¹H–¹⁵N shift correlation (HSQC) spectrum and the least accumulation of precipitate over time was 95% H₂O, 5% ²H₂O, pH 2.9, and 37 °C. Through analysis of standard triple resonance data sets with 0.2–0.3 mm ¹³C,¹⁵N samples under these conditions, we were able to assign the backbone of all residues, except Ile-105 (Fig. 2A).

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Figure 2.

Assignment and secondary structure of human TGF-β2. A, assigned ¹H-¹⁵N HSQC spectrum of TGF-β2 recorded in 5% ²H₂O, pH 2.7, 37 °C, 700 MHz. Assigned backbone amide signals are indicated by their residue number and one-letter code. Horizontal bars identify the side-chain amide resonances of Asn and Gln. B, secondary structure probabilities of TGF-β2 calculated using the program PECAN (67), with helical and strand probabilities plotted as positive and negative values, respectively. Secondary structure shown along the top was calculated from the crystal structure of TGF-β2 (PDB 2TGI) (68) using the program DSSP (69).

The secondary structure propensities, calculated from the assigned shifts using the program PECAN, showed that with the exception of the heel helix (α3), the protein had the expected secondary structure (Fig. 2B). The absence of a detectable heel helix suggests that rather than adopting a canonical closed form in which the two monomers pack extensively against one another, as in the two reported TGF-β2 crystal structures (40, 41), the protein instead adopts the open form, i.e. an alternative conformation, previously reported for TGF-β3 (42), in which the monomers rotate away from one another and the heel helix unfolds (Fig. S2).

TGF-β2/BG_ZP-C complex at high pH

To investigate possible conditions for studying the TGF-β2/BG_ZP-C complex, samples of 10 μm TGF-β2 with a slight molar excess of BG_ZP-C (2.25 eq for each 1.0 eq of TGF-β2 homodimer) were prepared between pH 3.0 and 11.0 in 1.0 pH increments. This analysis showed that all samples had extensive precipitate, except for the pH 10.0 sample, which was slightly precipitated, and for the pH 11.0 sample, which had no precipitate.

To determine whether the TGF-β2/(BG_ZP-C)₂ complex was stably formed at pH 11.0, Ile, Leu, Val-methyl–protonated but otherwise fully deuterated TGF-β2 and BG_ZP-C samples (²H–ILV–TGF-β2 and ²H-ILV-BG_ZP-C, respectively) were prepared by expression in Escherichia coli on the appropriately isotopically labeled medium, refolded, and examined alone by NMR at high pH, 25 mm [²H]glycine, pH 11.0, and at lower pH values, where we expected them to be soluble and natively folded (Fig. S3, A and B). The methyl spectra revealed some shift perturbations between the two pH values, especially for TGF-β2, yet regardless of the pH, the spectra were generally well-dispersed, suggesting that both TGF-β2 and BG_ZP-C are mostly, or entirely, natively folded at pH 11. To determine whether TGF-β2 and BG_ZP-C could still bind under these conditions, SPR sensorgrams were recorded as BG_ZP-C was injected over minimally-biotinylated TGF-β2 captured on a high-density streptavidin surface at pH 7.4 and pH 11.0 (Fig. S3, C and D). The K_D values at the two pH values, derived by fits of the equilibrium response as a function of concentration to a standard binding isotherm (Fig. S3, C and D), showed that there was only a 3-fold weakening of the affinity at pH 11 compared with pH 7.4 (Table 1). Thus, both TGF-β2 and BG_ZP-C appear to retain most or all of their native structure at pH 11.0 and in turn bind one another with an affinity that is only slightly weakened relative to that at neutral pH.

TGF-β2 pH titration

To obtain the TGF-β2 Ile, Leu, and Val (ILV) methyl signal assignments at pH 11.0 where we intended to map the BG_ZP-C–binding site, standard NMR experiments, such as C(CO)NH, H(CCO)NH, and HCCH-TOCSY, were applied to samples of ¹³C,¹⁵N TGF-β2 at pH 2.9 to extend the assignments from the backbone to the ILV side-chain methyls. This enabled the identification of the methyl signals of all the ILV residues in TGF-β2. To verify the assignments, a ¹H-¹³C shift correlation spectrum of ²H–ILV–TGF-β2 was recorded under the same conditions and overlaid onto the constant time ¹H-¹³C shift correlation spectrum of uniformly ¹³C,¹⁵N-labeled TGF-β2 (Fig. 3A). The 22 signals apparent in the spectrum of ²H–ILV–TGF-β2, which was expected based on the 7 Ile, 11 Leu, and 4 Val residues in TGF-β2 and the methyl precursors used, 3,3′-²H, 4-¹³C α-ketobutyrate for Ile (43) and 2-¹³C-methyl-2-hydroxy-3-oxobutanoate for Leu and Val (44), overlaid perfectly with the seven assigned Ile-δ1 signals and one of two methyls for the 11 assigned Leu-δ1/δ2 and four assigned Val γ1/γ2 signals.

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Figure 3.

pH titration of human TGF-β2. A, overlay of the methyl region of the CT-HSQC spectrum of ¹³C,¹⁵N-TGF-β2 (red contours) or HSQC spectrum of ²H-MP-TGF-β2 (blue contours). Spectra were recorded at pH 2.7 and 37 °C. Assigned methyls are indicated by their residue number and one-letter code. B, HSQC spectra of ²H-MP-TGF-β2, recorded between pH 2.7 and 4.9. Overlay of the end points of the titration at pH 2.7 (blue) and pH 4.9 (red) is shown in the main panel. Peaks assignments shown correspond to those that could be confidently inferred from the assigned signals at pH 2.7. Expansions of three representative peaks, Ile-22, Ile-33, and Ile-105, at all points along the titration, are shown in the inset. C, overlay of the methyl region of the HSQC spectrum of ²H-MP-TGF-β2 at pH 4.9 (red contours) and 11.0 (black contours). Peaks assignments shown correspond to those that could be confidently inferred from the assigned signals at pH 2.7.

To transfer the methyl assignments from pH 2.7 to pH 11.0, ¹H-¹³C shift correlation spectra of ²H–ILV–TGF-β2 were collected under the limited range of pH values, 2.7–4.9 and 11.0, where 2 μm or higher samples could be prepared without significant precipitation. This showed unexpectedly large shift changes between pH 2.7 and 4.9 but surprisingly small changes between pH 4.9 and 11.0 (Fig. 3, B and C). Although the spectra in the low pH range were collected in relatively fine increments, it nonetheless proved difficult to follow all but the most resolved signals as the majority of signals exhibited slow exchange behavior, with signals corresponding to low and high pH forms of the protein being simultaneously observed at intermediate pH values in this range (Fig. 3B, insets). This behavior suggested that the large shifts observed as the pH was changed from 2.7 to 4.9 were not due to ionization of nearby side-chain carboxylic acid groups but instead a large rearrangement of the protein. This slow exchange behavior is also accompanied by an apparent shift of several methyl signals from the random coil methyl position (∼0.85 ppm ¹H for Ile-δ1 and Leu-δ methyls and ∼0.90 ppm ¹H for Val-γ methyls) to a nonrandom coil position as the pH was raised from 2.7 to 4.9. One process that could account for these observations is a large-scale conformational rearrangement from the open form at pH 2.7 to the closed form at pH 4.9. The slow exchange behavior is presumably caused by the opening and closing process, which would be expected to be relatively slow (millisecond time scale) owing to the large number of atoms that must simultaneously rearrange. The decrease in the number of random coil methyls is presumably due to the 1 Val (Val-61), 2 Leu (Leu-62 and Leu-64), and 1 Ile (Ile-68) residues in the heel helix that are structurally disordered in the open form but not the closed form.

Although the slow exchange behavior prevents reliable assignment of most of the methyl signals, it is nonetheless possible to tentatively assign a few of the peaks at pH 11.0, such as Leu-89 or Leu-101, which did not appear to shift at all, or Ile-33, Ile-22, Ile-88, and Ile-105, which shifted as the pH was varied, but were well-resolved (Fig. 3, A–C). Thus, due to the unexpected large-scale conformational rearrangement and slow exchange behavior, it was possible to transfer only about a third of the assigned methyl signals at pH 2.7 to pH 11.0.

Titration of TGF-β2 with BG_ZP-C

To identify the BG_ZP-C–binding site on TGF-β2, ¹H-¹³C shift correlation spectra of ILV–TGF-β2 were collected at pH 11 with sub- to super-stoichiometric amounts of unlabeled BG_ZP-C added (Fig. 4, A and B). Through spectral overlays, it was evident that three of the tentatively assigned signals, Ile-33, Ile-88, and Leu-101, underwent sizable shifts. These three residues are each located on the concave inner surface of the extended finger region and are spatially quite close to one another, with the γ2 methyl of Ile-33 packing against the Ile-88 δ1 methyl and the Leu-101 δ2 methyl packing against the Ile-88 δ2 methyl (Fig. 5A). The other three tentatively assigned methyls that did not shift, Ile-22, Ile-105, and Leu-89 (Fig. 4A), were not spatially close to one another, with two them, Leu-89 and Ile-105, lying far apart on the convex outer surface of the extended finger region, and one of them, Ile-22, buried in the hydrophobic interface between monomers (Fig. 5A). This strongly suggested that the BG_ZP-C–binding site on TGF-β2 lies principally on the concave inner surface of the fingers.

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Figure 4.

Titration of ²H-MP human TGF-β2 with unlabeled BG_ZP-C. A, overlay of the methyl region of the HSQC spectrum of ²H-MP-TGF-β2 in either the unbound state (blue contours) or bound state (red contours) with an excess of unlabeled BG_ZP-C (1 eq of TGF-β2 homodimer with 2.25 eq of BG_ZP-C). Peak assignments shown correspond to those that could be confidently inferred from the assigned signals at pH 2.7. B, expansions of six representative peaks, Ile-33, Ile-88, and Leu-101, and three other unassigned peaks, designated peaks a–c, at all points along the titration. C, overlay of the HSQC spectrum of WT ²H-MP-TGF-β2 (blue contours) and the I33L variant (red contours). Inferred assignments for Ile-33 in the WT protein and Leu-33 in the variant are shown. D, overlay of the HSQC spectrum of WT ²H-MP-TGF-β2 (blue contours) and the L101I variant (red contours). Inferred assignments for Leu-101 in the WT protein and Ile-101 in the variant are shown.

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Figure 5.

Structure of human TGF-β2 homodimer and identification of residues in the BG_ZP-C–binding site. A, structure of the TGF-β2 homodimer (PDB 2TGI), highlighting the Ile, Leu, and Val residues found on either the inside or outside of the extended finger region (red and blue text, respectively). Same residues are shown on only one of the two monomers to simplify the presentation. B, structure of the TGF-β2 homodimer (PDB 2TGI), highlighting the residues that lie on either the inside or outside of the fingers that were substituted in this study (red and blue text, respectively). Same residues are shown on only one of the two monomers to simplify the presentation. C, structure of the engineered TGF-β mini-monomer, mmTGF-β2-7M (PDB 5TX6), highlighting the Ile, Leu, and Val residues found on the inside of the extended finger region (red text). Engineered loop that replaced the heel helix is highlighted in green. D, alignment of the residues from the finger region of all TGF-β family growth factors in humans; positions highlighted in color were either shown to shift upon titration of ²H-MP-TGF-β2 with unlabeled BG_ZP-C or to be affected in their binding affinity for BG_ZP-C upon substitution. Hydrophobic residues are colored green; acidic residues are colored red; basic residues are colored blue, and neutral residues are colored purple. Residues highlighted in yellow correspond to those that are either entirely (Lys-97) or mostly unique (Val-92/Ile-92 and Glu-99) to TGF-βs and Inh α.

To ascertain whether this was correct, two separate ²H–ILV–TGF-β2 samples were prepared in which Ile-33 and Leu-101 were conservatively substituted with leucine and isoleucine, respectively (I33L and L101I). The ¹H-¹³C shift correlation spectra of these variants were then recorded at pH 11.0 and compared with the spectrum of WT ²H–ILV–TGF-β2 (Fig. 4, C and D). The spectra of I33L ²H–ILV–TGF-β2 and L101I ²H–ILV–TGF-β2 each had no peak corresponding to the assigned Ile-33 and Leu-101 peaks, respectively, while at the same time they both had an extra peak, located in the Leu/Val region of the spectrum for the I33L variant and in the Ile region of the spectrum for the L101I variant. This indicates the assignments for the Ile-33 and Leu-101 methyls were correct. One further point to note is that the peak tentatively assigned to Ile-88 in the spectra of both the I33L and L101I variants also shifted. This is expected even with these conservative Ile → Leu and Leu → Ile substitutions, owing to the close spatial proximity of these residues, and thus it provides further, albeit indirect, evidence that all three of these assignments are correct.

The overlay of the spectrum of ILV–TGF-β2 bound to an excess of BG_ZP-C at pH 11 relative to ILV–TGF-β2 alone revealed that several other as yet unassigned methyls also underwent sizable shifts upon addition of BG_ZP-C (Fig. 4A). If the binding site indeed lies on the concave inner surface of the fingers, then it is reasonable to expect that the unassigned peaks that underwent the largest shifts (Fig. 4A, peaks a–d) correspond to other methyl-bearing residues, such as Leu-28, Val-61, Leu-64, Leu-86, and Ile-92, that are spatially close to Ile-33, Ile-88, and Leu-101 (Fig. 5A). To determine whether this was correct, four additional conservatively substituted ILV–TGF-β2 variants were prepared, L28I, V61I, L86I, and I92L. The ¹H-¹³C shift correlation spectra of these variants, relative to that of WT ILV–TGF-β2, confirms this, with peaks a–d in the WT spectrum corresponding to Ile-92, Val-61, Leu-28, and Leu-86, respectively (Fig. S4).

Together, these results show that the BG_ZP-C–binding site lies on the concave inner surface of the fingers and extends horizontally from the near the loops connecting the fingertips toward the edge of the heel helix and vertically from finger 1 at the bottom to finger 4 at the top (Fig. 5A). The binding site overlaps in part with the TβRII-binding site, which includes the loops connecting fingers 1/2 and 3/4, but it does not appear to extend to the convex outer surface of the fingers since the signals of two assigned residues in this region, Leu-89 and Ile-105, did not shift upon addition of BG_ZP-C (Fig. 4A). Another observation in accord with this is that Ile-98, which lies on the outside of the fingers (Fig. 5A) and by a process of elimination must correspond to one of the two unassigned Ile resonances between 0.65–0.75 ¹H and 13.0–13.5 ppm ¹³C (Fig. 4A), does not shift upon addition of BG_ZP-C (Fig. 4A).

Site-directed mutagenesis of TGF-β2 and effects on BG_ZP-C and BG_O binding

To further validate the BG_ZP-C–binding site and to identify potential interaction hot spots, several residues both inside the binding site on the concave surface of the fingers and outside on the convex surface were substituted with alanine. The residues on the inside that were substituted include the three residues whose methyl groups shifted the most upon BG_ZP-C binding, Ile-33, Ile-92, and Leu-101, and three other prominent nonmethyl-bearing residues, Tyr-90, Lys-97, and Glu-99 (Fig. 5B). The residues on the outside that were substituted include two residues whose methyl groups did not appreciably shift, Leu-89 and Ile-98, and three other prominent residues, Glu-84, Thr-87, and Tyr-91 (Fig. 5B).

Although it was possible to isolate each of these variants in high yield from inclusion bodies, the amount of dimer that could be isolated following the folding reaction varied significantly, with some variants, such as I33A, leading to reduced but still significant dimer formation, whereas others, such as I98A and Y90A, led to little to no dimer (Fig. S5). The criterion whether a variant could be purified was whether subsequent fractionation of the dimeric form of the protein on a C18 reverse-phase column resulted in a suitable single peak with LC-ESI-MS intact mass within 0.5 Da of that expected. Those variants that did not meet this criterion, Y90A and L101A, were excluded from further analysis. The SPR measurements were performed by capturing minimally biotinylated TGF-β2 dimers onto a streptavidin surface, followed by duplicate or triplicate injections of either BG_ZP-C or BG_O until equilibrium was reached. Typical sensorgrams, and accompanying fits of the equilibrium response as a function of concentration, are presented in Fig. 6. The measured binding constants are presented in Table 1.

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Figure 6.

SPR-based binding studies of mouse TGF-β2 and variants. A, SPR sensorgrams for injection of a 2-fold dilution series of BG_ZP-C (2 μm to 3.9 nm) or BG_O (2 μm to 3.9 nm) over immobilized WT TGF-β2 (left and right panels, respectively). Injections were performed in duplicate over the time period shown by the solid gray bar. B, saturation plots in which the equilibrium response from the sensorgrams in A were fit as a function of the injected concentration to a standard binding isotherm to derive the K_D and the R_max. Fitted values and the estimated errors are provided in Table 1. C and E, sensorgrams as in A, but for BG_ZP-C (2 μm to 3.9 nm) or BG_O (2 μm to 3.9 nm) injected over immobilized TGF-β2 I33A or TGF-β2 T87A, respectively. D and F, saturation plots as in B, but for TGF-β2 I33A or TGF-β2 T87A, respectively.

The substitution of the four residues on the inside of the fingers with alanine, Ile-33, Ile-92, Lys-97, and Glu-99, each led to a 5-fold or greater decrease in the binding affinity for BG_ZP-C, with substitution of Ile-33, whose methyl group was strongly perturbed, leading to the largest alteration, 41-fold. The substitution of the five residues on the outside of the fingers with alanine, Glu-84, Thr-87, Leu-89, Tyr-91, and Ile-98, led in contrast to at most a 2-fold weakening of the binding affinity for BG_ZP-C. To ascertain whether the substitutions that led to a large disruption of BG_ZP-C–binding affinity did so indirectly, the binding affinity of all nine alanine variants for BG_O, which has been shown previously to bind at a site distinct from BG_ZP-C (17), were also measured. The BG_O–binding affinities of all the TGF-β2 alanine variants were within 1.5-fold that of WT TGF-β2, except TGF-β2 Y91A, which was within 2.6-fold. These results show that the large decrease in the binding affinity for BG_ZP-C by the four variants on the inside of the fingers cannot be explained by indirect effects, such as the presence of non-native protein or an overall perturbation of the structure, but instead it must be due to loss of specific contacts that contribute favorably to binding.

Binding of BG_ZP-C to mini-monomeric TGF-β

To further confirm and narrow down the possible BG_ZP-C–binding site, we took advantage of an engineered TGF-β monomer previously reported (36), designated mmTGF-β2-7M, that included the entire finger region but had a flexible loop in place of the heel helix (Fig. 5C). This form of TGF-β, which was based on the backbone of TGF-β2, also included several substitutions from TGF-β1, including I92V, to confer high-affinity TβRII binding. To investigate whether mmTGF-β2-7M might bind BG_ZP-C, we captured a biotinylated avi-tagged form of the protein on a high-density SPR streptavidin surface, measured its affinity to BG_ZP-C by equilibrium analysis, and found that the K_D values were weakened by roughly 10-fold relative to WT TGF-β2 dimer at pH 7.4 (K_D 2.1 μm versus 0.20 μm, Table 1). This indicated that mmTGF-β2-7M indeed possessed most, but not all, of the residues that form the BG_ZP-C–binding site.

To further investigate the binding, we prepared deuterated ILV-methyl–protonated mmTGF-β2-7M and extended the assignments from the backbone to the side-chain methyl groups under conditions where the protein was assigned (10 mm sodium phosphate, 10 mm CHAPS, pH 4.7). To transfer the methyl assignments to the conditions for a titration experiment, pH 11 in the absence of CHAPS, we collected spectra at a constant concentration of CHAPS (10 mm) as the pH was raised to 11, and at pH 11, with a descending concentration of CHAPS. These titrations led to small shift perturbations for most residues and were in the fast exchange regime, thus enabling assignment of all 18 expected ILV methyl resonances at pH 11 in the absence of CHAPS (Fig. S6A).

The addition of BG_ZP-C to ILV-mmTGF-β2-7M at pH 11.0 led to sizable shifts and slow exchange binding for several residues, including Ile-33, Ile-88, Leu-28, and Leu-86 (Fig. S6A). Val-92 and Leu-101 also shifted based on the disappearance of their unbound signals when more than 1.0 eq of BG_ZP-C had been added (Fig. S6, A and B). The extent of these shifts was, however, unclear, as it was impossible to unambiguously identify the corresponding bound signals due to the relatively close proximity of the unbound and bound signals to one another. The signals that were perturbed in mmTGF-β2-7M by BG_ZP-C were all the same (or homologous) to those in TGF-β2, except Val-61, which was located in the heel helix and was absent in mmTGF-β2-7M. Together, the SPR and NMR results show that mmTGF-β2-7M binds BG_ZP-C in the same overall manner as TGF-β2, albeit with an affinity that is about 10-fold weaker. The weakening is probably due to lack of contact with Val-61 and other surrounding residues in the heel helix, which are absent in mmTGF-β2-7M.

Expression and purification of TGF-β growth factor domains

The growth factor domains of human TGF-β2, mouse TGF-β2, and accompanying variants, the previously reported TGF-β2 mini-monomer, mmTGF-β2–7M (36), and N-terminally avi-tagged mmTGF-β2-7M were each expressed in Escherichia coli at 37 °C in the form of insoluble inclusion bodies (sequences of the expressed proteins are provided in Fig. S7). The cells were disrupted by sonication, and after washing, the overexpressed protein was reconstituted in 8 m urea and refolded by dilution into nondenaturing buffer at 4 °C containing 30 mm CHAPS (Gold Biotechnology, Denver, CO) at pH 9.5. The natively folded growth factor domains were isolated by high-resolution cation–exchange chromatography (Source S, GE Healthcare), followed by reverse-phase chromatography (C18 Jupiter, Phenomenex, Torrance, CA). Intact masses of the purified proteins were measured by LC electrospray ionization TOF MS (LC-ESI-TOF-MS, Bruker Micro TOF, Billerica, MA) and were found in all cases to be within 0.5 Da of their calculated masses. Other details can be found in the previously described protocol for expressing, refolding, and purifying TGF-β2 (52).

Expression and purification of mammalian-expressed rBG_O-ZP, rBG_O, and rBG_ZP-C

Rat betaglycan extracellular domain, rBG_O-ZP, and its two component TGF-β–binding domains, the orphan domain, rBG_O, and the C-terminal zona pellucida domain, rBG_ZP-C, were each expressed as secreted proteins in suspension-cultured HEK-293 expi cells and HEK-293 expi expression medium (GE Healthcare), as described previously (29). Secreted proteins were purified from the conditioned medium by performing a 2-fold dilution into PBS, followed by sequential purification of the C-terminally His-tagged proteins by nickel metal-affinity chromatography (chelating Sepharose, GE Healthcare) and size-exclusion chromatography (Superdex 200, GE Healthcare), as described previously (29).

Expression and purification of bacterially-expressed TβRII and rBG_ZP-C

Unlabeled TβRII ectodomain, for use in the biotinylation reactions, was produced as an insoluble protein in E. coli and refolded and purified as described previously (52). Isotopically labeled samples of rBG_ZP-C for NMR were produced in E. coli by inserting the coding region for the C-terminal portion of the rat betaglycan zona pellucida domain (residues 590–756 of rat betaglycan, with residue numbering beginning at the N-terminal methionine) downstream of the coding region for thioredoxin (Trx), a hexahistidine tag, and a thrombin cleavage site, LVPRGS, in the bacterial T7 expression vector pET32a (EMD Millipore, Billerica, MA). The Trx–BG_ZP-C fusion protein was overexpressed in BL21(DE3) cells cultured in LB medium at 37 °C containing 50 μg/ml carbenicillin. Cells were resuspended in 100 mm Tris, 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, pH 8.0, and lysed by sonication. Inclusion bodies were isolated by washing the insoluble fraction with lysis buffer with 500 mm NaCl, followed by lysis buffer with 1% Triton X-100. Inclusion bodies were solubilized by stirring for 12 h in 8 m urea, 25 mm Tris, 50 mm DTT, pH 8, and after removal of the remaining insoluble material by centrifugation, the supernatant was buffer exchanged into 8 m urea, 25 mm Tris, pH 4. Urea solubilized Trx–BG_ZP-C was then slowly diluted into folding buffer (100 mm Tris, 0.25 m guanidine hydrochloride, 0.5 m arginine, 2 mm GSH, 0.5 mm GSSG, pH 9.0) to a final protein concentration no higher than 0.1 mg/ml. After stirring 14 h at 4 °C, the folding mixture was concentrated and dialyzed into purification buffer (2 m urea, 10 mm Tris, 15 mm CHES, 1 μm pepstatin, 1 μm leupeptin, 1 mm benzamidine, pH 9.2). After centrifugation and filtration, Trx–BG_ZP-c was fractionated by loading it onto a Source Q anion-exchange column (GE Healthcare) equilibrated with purification buffer and eluted with a linear gradient from 0 to 0.3 m NaCl. After overnight digestion with thrombin (3 units/mg fusion protein), the cleavage mixture was loaded onto a nickel-affinity column (chelating Sepharose, GE Healthcare) equilibrated in 25 mm sodium phosphate, 200 mm NaCl, pH 8.0, which enabled separation of rBG_ZP-C away from His-tagged thioredoxin. Intact mass of the purified protein was measured by electrospray ionization TOF MS (Bruker Micro TOF, Billerica, MA) and was found to be within 1.0 Da of its calculated mass.

Expression of deuterated, methyl-protonated proteins

Deuterated Ile, Leu, and Val methyl-protonated human TGF-β2 and mmTGF-β2–7M (²H-MP-TGF-β2 and ²H-MP-mmTGF-β2-7M) were produced by inoculating colonies from a freshly transformed plate of BL21(DE3) cells into water-based M9 medium followed by incubation at 37 °C. Once the cell density rose to 0.25, the cells were pelleted in a sterile centrifuge bottle and inoculated into the same volume of ²H₂O-based M9 medium containing 99.9% ²H₂O and 3 g/liter [²H]glucose (Cambridge Isotope Laboratories, Andover, MA) and incubated at 37 °C. Once the cell density reached 0.55, 75 mg of 4-¹³C-3,3′-²H-α-ketobutyric acid (Cambridge Isotope Laboratories, Andover, MA) and 200 mg of saponified ethyl 2-hydroxy-2-¹³C-methyl-3-oxobutanoate were added (saponification was achieved by incubating 200 mg of ethyl 2-hydroxy-2-¹³C-methyl-3-oxobutanoate for 1 h at 25 °C in 10 ml of ²H₂O containing 0.1 m sodium hydroxide). Protein expression was induced 1 h later by the addition of 200 mg IPTG.

Backbone assignment of TGF-β2

Samples of ¹³C,¹⁵N human TGF-β2 for backbone assignment were prepared in 5% ²H₂O at pH 2.7 at a protein concentration of 0.25 mm - 0.35 mm in a 5 mm Shigemi tube (Shigemi, Allison Park, PA). All NMR data were acquired at 37 °C using a Bruker AVI 700 MHz spectrometer (Bruker, Billerica, MA) equipped with 5 mm ¹H{¹³C,¹⁵N} TCI cryogenically cooled probe. Backbone resonance assignments of TGF-β2 were obtained by collecting and analyzing sensitivity-enhanced triple-resonance data sets, including HNCACB (53), CBCA(CO)NH (54), HNCA (55), HN(CO)CA (55), HNCO (56), and HN(CA)CO (57). All spectra were processed with NMRPipe (58) and analyzed using NMRViewJ (59).

Methyl spectra

Samples of ²H–ILV–TGF-β2 or ²H-ILV-mmTGF-β2-7M for the binding titrations were prepared in 25 mm 2H-glycine at pH 11.0 in either straight 3 mm tubes (Wilmad, Vineland, NJ) or a 5 mm susceptibility matched microcells (Shigemi, Allison Park, PA). Separate samples, each with the labeled protein being 15 - 25 μm, were used for each point in the titration. Spectra were recorded at 37 °C using a ¹H-¹³C HSQC pulse sequence with a WATERGATE solvent suppression scheme (60) as implemented on a Bruker AVII 800 MHz spectrometer (Bruker, Billerica, MA) equipped with a 5 mm ¹H{¹³C,¹⁵N} TCI cryogenically cooled probe. Spectra were acquired with 128 complex points and between 64 and 128 scans per time increment. Spectra were processed with NMRPipe (58) and analyzed using NMRFAM-SPARKY (61) and CcpNmr (62). Spectra of ²H-MP TGF-β2 for the pH titrations were acquired in the same manner, except samples were prepared in 5% ²H₂O with the pH being adjusted by the addition of small aliquots of 0.1 m NaOH or 0.1 m HCl prior to NMR data collection.

GF biotinylation for SPR

Minimally biotinylated mouse TGF-β2, and single amino variants, were prepared by overnight dialysis of a 0.5 ml solution containing 125 μg growth factor homodimer (5 nmol) and 200 μg TβRII (15 nmol) into 25 mm MES, pH 6.0. DMSO was added to a final concentration of 40% v/v, followed by 0.0115 mg of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Pierce) (60 nmol), 0.026 mg of sulfo-N-hydroxysulfosuccinimide (120 nmol, Pierce), and 0.20 mg (+)-biotinyl-3,6,9-trioxaundecanediamine (EZ-Link Amine-PEG3-Biotin) (480 nmol, Pierce). The reaction was allowed to proceed for 1 h at 25 °C and then quenched by the addition of 0.4 ml 100 mm acetic acid. The growth factor was separated away from TβRII and the reagents by loading it onto a Source S high resolution cation exchange column equilibrated in 20 mm sodium acetate, 30% isopropanol pH 5.2 and eluting it with a linear gradient from 0 - 70% B over 10 column volumes. LC-ESI-TOF MS showed that roughly 10% of the growth factor was singly modified, while the remaining 90% was unmodified, indicating that at most the growth factor was singly biotinylated.

Biotinylated avi-tagged mmTGF-β2–7m was prepared by overnight dialysis of 0.5 mg of avi-tagged mmTGF-β2–7m and 1.0 mg TβRII into 25 mm Bicine, pH 8.3. Biotinylation was achieved by incubation at 37 °C for 4 h in the presence of 40 μm biotin, 10 mm ATP, 10 mm Mg(OAc)₂, and 20 μg of BirA biotin ligase (63). Biotinylated avi-tagged mmTGF-β2–7m was isolated from the reaction mixture as above for chemically biotinylated TGF-β2 homodimers.

SPR measurements

SPR binding studies were performed with a BIAcore 3000 instrument (GE Healthcare, Piscataway, NJ) and were analyzed using the software package Scrubber2 (Biologic Software, Canberra, Australia). Streptavidin-coated sensor chips for capture of biotinylated ligands were made by activating the surface of a CM-5 sensor chip (GE Healthcare) with EDC and N-hydroxysulfosuccinimide (NHS) (Pierce) followed by injection of streptavidin (Pierce) diluted into sodium acetate at pH 4.5 until the surface density reached 6000 - 8000 RUs. Biotinylated ligands were captured onto the streptavidin surface to a surface density between 50–300 RU. Equilibrium binding assays were performed by injecting the analytes, BG_ZP-C or BG_O, in 10 mm HEPES, 150 mm NaCl, 0.1% surfactant P20 (Pierce) at pH 7.4 (HBS-EP buffer) at a rate of 10 μl min⁻¹ for 720 s, followed by a dissociation period of 600 s. Regeneration of the surface was achieved by a 10 s injection at 100 μl min⁻¹ of 4 m guanidine hydrochloride in 4 mm HCl solution. Baseline correction was performed by double referencing. Equilibrium analyses were performed by fitting the equilibrium to the equation RU = RU_max·Conc/(K_D + Conc). Binding studies of BG_O-ZP and noggin were performed as described above, but with human BMP-2 and human TGF-β2 directly attached to the surface of a CM5 sensor chip by amine coupling with NHS and EDC. Human BMP-2 and noggin for these measurements were purchased from PeproTech (Rocky Hill, NJ).

David Brinkman is thanked for the initial efforts to generate and characterize several TGF-β2 variants; Erich Hellemann for measuring the mass of the TGF-β2 variants; Mike Delk for maintaining the NMR instruments, and Profs. Craig Harrison and Daniel Bernard for providing valuable comments on the manuscript. Molecular graphics and analyses were performed with UCSF Chimera. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health NIGMS Grant P41-GM103311).