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

Abstract

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-β family that selectively binds transforming growth factor-β (TGF-β) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo Here, using NMR titrations of methyl-labeled TGF-β2 with BG's C-terminal binding domain, BG_ZP-C, and surface plasmon resonance binding measurements with TGF-β2 variants, we found that the BG_ZP-C-binding site on TGF-β2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-β isoforms and the InhA α-subunit, but they are unconserved in other TGF-β family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-β2 variants with the corresponding residues from BMP-2 bound BG_ZP-C more weakly than corresponding alanine variants. The BG_ZP-C-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-β2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-β2 and InhA bind BG_ZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BG_ZP-C-binding site on TGF-β2 and shed light on the specificity of BG for select TGF-β-type GFs and the mechanisms by which BG influences their signaling.

Keywords: ILV methyl labeling; betaglycan; cardiac development; cell signaling; cell surface receptor; endocrinology; finger region; nuclear magnetic resonance (NMR); transforming growth factor β (TGF-B).

Figure 1.

Overall domain structure of betaglycan and proposed mechanisms for potentiation of TGF-β signaling and inhibition of activin signaling. A, overall domain structure of betaglycan. B, previously proposed mechanism for potentiation of TGF-β signaling by betaglycan (29) in which BG binds TGF-β dimers asymmetrically with a 1:1 stoichiometry in a manner that blocks one of the TβRII-binding sites but not the other (step i). Sequestration of TGF-β2 on the membrane by BG is proposed to promote binding of TβRII (step ii), which in turn promotes the concerted recruitment of TβRI and displacement of the BG orphan domain, and by an unknown mechanism, binding of a second TβRI:TβRII pair (step iii). C, previously proposed mechanism for inhibition of activin signaling by InhA (21, 23) in which BG binds InhA through its α-subunit (step i). Sequestration of InhA on the membrane by BG is proposed to promote binding of ActRII, which in turn sequesters ActRII away from activin A in a complex that is incapable of recruiting ActRIb and signaling (step ii).

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).

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.

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.

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.

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.

Figure 7.

SPR-based binding studies of BMP-2. A and C, SPR sensorgrams for injection of BG_O-ZP (BG) over immobilized BMP-2 (A) or TGF-β2 (C) in HBS-EP buffer adjusted to pH 7.4. Raw sensorgrams, recorded in duplicate, are shown in gray, and the fitted curves (TGF-β2 only) for the 1:1 binding model are shown in orange. Concentrations of BG injected over the BMP-2 and TGF-β2 surfaces were 12.5, 25, 50, and 100 nm and 25, 50, and 100 nm, respectively. B, SPR sensorgrams for injection of noggin over immobilized BMP-2 in HBS-EP buffer adjusted to pH 7.4. Concentrations of noggin injected were 1.95, 3.9, 7.8, 15.6, 31.2, 62.5, 125, and 250 nm.