An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

Abstract

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a K_i of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.

Keywords: cancer; cell signaling; dominant negative; fibrosis; inhibitor; protein engineering; transforming growth factor beta (TGF-B).

Figure 1.

Structure of the TGF-β signaling complex and sequences of the engineered TGF-β variants lacking the heel helix, α3. A, schematic representation of the TGF-β signaling complex formed between human TGF-β3 homodimer (magenta and blue ribbons) and the extracellular ligand binding domains of the human TGF-β type I and type II receptors, TβRI (red ribbon) and TβRII (tan ribbon) (PDB 2PJY) (8). The disulfide bonds, including the single inter-chain disulfide connecting the TGF-β monomers, are depicted in yellow. The TGF-β monomers are described as curled left hands, with the heel formed by a 3½ turn α-helix (α3) and the four fingers formed by the β-strands that extend from the cystine knot that stabilizes each monomer. B, expanded view illustrating packing interactions formed by hydrophobic residues that emanate from the heel α-helix (blue ribbon) of one TGF-β3 monomer with hydrophobic residues from the palm region of the opposing TGF-β3 monomer (magenta ribbon with transparent magenta surface). C, expanded view illustrating ionic, hydrogen bonding, and hydrophobic interactions that stabilize TβRI (red ribbon) at the composite interface formed by both monomers of TGF-β3 (magenta and blue ribbons) and TβRII (tan ribbon). D, sequence alignment of TGF-β1, -β2, and -β3 with monomeric variants in which Cys-77, which normally forms the inter-chain disulfide bond, is substituted with serine (mTGF-β2 and mTGF-β3) or mini-monomeric variants in which Cys-77 is substituted with serine, residues 52–71 have been deleted, and two or three additional residues (highlighted in red) have been substituted (mmTGF-β1, mmTGF-β2, and mmTGF-β3). Calculated net charge of the corresponding monomers at pH 7.0 is shown on the right. E, sequence alignment of TGF-β1, -β3, -β2, mmTGF-β2, and mmTGF-β2-7M in the TβRII-binding region. Residues in the TβRII binding interface are indicated by yellow shading. Residues substituted in mmTGF-β2-7M relative to mmTGF-β2 are highlighted in red, and include K25R, I92V, and N94R, which were shown previously to be necessary and sufficient for high affinity TβRII binding (39, 40). F, interface between TGF-β3 and TβRII, with Arg-25, Val-92, and Arg-94 highlighted by red labels.

Figure 2.

Structure of mmTGF-β2. A, assigned ¹H-¹⁵N HSQC spectrum of mmTGF-β2 recorded in 10 mm sodium phosphate, 10 mm CHAPS, 5% ²H₂O, pH 4.7, 37 °C, 800 MHz. Assigned backbone amide signals are indicated by their residue number and one-letter amino acid code. B, overlay of 1.8 Å crystal structure of mmTGF-β2 (orange ribbon) with one of the monomers from the 1.8 Å crystal structure of TGF-β2 (PDB 2TGI, blue ribbon). Major structural features are indicated, along with the newly created loop in mmTGF-β2 (red), which takes the place of the heel (α3) helix in TGF-β2. C, overlay of the two mmTGF-β2 chains (chain A and B shown in orange and green ribbon, respectively) from the crystallographic asymmetric unit. Other details as in B. D, overlay of mmTGF-β2 and TGF-β2 as in B, but with the aligned positions restricted to the residues 18–45 and 61–87 in fingers 1/2 and 3/4, respectively.

Figure 3.

Binding properties of mmTGF-β2 and mmTGF-β2-7M. A and B, SPR sensorgrams for injection of a 2-fold dilution series from 3 to 0.047 μm TβRII over immobilized TGF-β2 (A) or mmTGF-β2 (B). Responses shown were normalized for the surface density of the immobilized TGF-βs. C–H, SPR sensorgrams for injection of a 2-fold dilution series from 3 to 0.012 μm TβRII (C and D), 1.024 to 0.008 μm TβRI (E and F), or 1.024 to 0.008 μm TβRI in the presence of 2 μm TβRII in both the running buffer and injected samples (G and H) over immobilized avi-TGF-β3 (C, E, and G) or avi-mmTGF-β2-7M (D, F, and H). Sensorgrams shown in C, D, and G were fitted to a 1:1 binding model; raw data are shown in black, and the fitted curve is shown in red. TGF-β2 and mmTGF-β2 were immobilized by direct carbodiimide-based amine coupling to the sensor surface, whereas avi-TGF-β3 or avi-mmTGF-β2-7M were immobilized by capturing the enzymatically biotinylated proteins onto the surface of sensor chip coated with streptavidin at high (∼8000 resonance units) density.

Figure 4.

Solubility of TGF-β2 and monomeric variants. A and C, TGF-β2 and mTGF-β2 (A) and mmTGF-β2 and mmTGF-β2-7M (C) were diluted from a concentrated stock in 100 mm acetic acid into either PBS at 7.4 (Neutral pH) or 100 mm acetic acid (Acidic pH), and the light scattering at 340 nm was measured. B and D. TGF-β2 and mTGF-β2 (B) and mmTGF-β2 and mmTGF-β2-7M (D) samples diluted into either PBS or 100 mm acetic acid were centrifuged for 5 min at 20,000 × g, and the protein absorbance at 280 nm was measured.

Figure 5.

Structure of mmTGF-β2-7M and mmTGF-β2-7M·TβRII complex. A, assigned ¹H-¹⁵N HSQC spectrum of mmTGF-β2-7M recorded in 10 mm sodium phosphate, 10 mm CHAPS, 5% ²H₂O, pH 4.70, 37 °C, 800 MHz. Assigned backbone amide signals are indicated by their residue number and one-letter amino acid code. B, overlay of 1.8 Å crystal structure of mmTGF-β2-7M (dark red ribbon) with one of the monomers from the 1.8 Å crystal structure of TGF-β2 (PDB 2TGI, blue ribbon). Major structural features are indicated, along with the newly created loop in mmTGF-β2 (red), which takes the place of the heel (α3) helix in TGF-β2. C, overlay of the three mmTGF-β2-7M chains (chain A, B, and C shown in dark red, green, and orange ribbon, respectively) from the crystallographic asymmetric unit. Dashed line corresponds to missing segments in the newly created loop in chains A and C due to weak electron density. Other details as in B. D, overlay of the 1.8 Å crystal structure of mmTGF-β2-7M·TβRII complex (dark red and orange ribbons, respectively) with one of the TGF-β3 monomers and its bound TβRII from the 3.0 Å crystal structure of the TGF-β3·TβRII·TβRI complex (PDB 2PJY, TGF-β3 monomer and TβRII shown in dark blue and cyan ribbon, respectively; TβRI not shown for clarity). Newly created loop in mmTGF-β2 (red), which takes the place of the heel (α3) helix in TGF-β2, is depicted in red. E, overlay as in B, but expanded to show the near identity of critical hydrophobic and hydrogen-bonding/electrostatic interactions shown previously to be essential for high affinity TGF-β3·TβRII binding (39, 40).

Figure 6.

Signaling activity of TGF-β dimers and monomers. A, TGF-β luciferase reporter activity for TGF-β1, mTGF-β3, and mmTGF-β2-7M shown in solid circles, squares, and triangles, respectively. The solid lines, colored red and blue, correspond to the fitted curves to derive the EC₅₀ (green line for mmTGF-β2-7M was not fit due to the lack of signaling activity for this variant). B, TGF-β luciferase reporter activity for cells treated with a sub-saturating concentration of TGF-β1 (8 pm) with increasing concentration of the indicated monomeric TGF-β variant added (mTGF-β3 and mmTGF-β2-7M shown in open squares and closed triangles, respectively). The solid blue line corresponds to the fitted curve for mTGF-β3 to derive the EC₅₀. The solid green line corresponds to the fitted curve for mTGF-β2-7M to derive the IC₅₀.

Figure 7.

TR-FRET assay for ligand-mediated assembly of TβRI·TβRII complexes. A, structure of the TGF-β3·TβRII·TβRI complex with tags appended to the C terminus of TβRI and TβRII and fluorescently labeled donor and acceptor proteins that associate with the tags. TβRII has a C-terminal hexahistidine tag (His₆) and is bound by an Tb³⁺-cryptate-labeled antihexahistidine tag antibody (CisBio, Bedford, MA). TβRI has a C-terminal biotinylated avitag and is bound by XL₆₆₅-labeled streptavidin (CisBio, Bedford, MA). The single lysine residue in the TβRI C-terminal avitag that is biotinylated is labeled as K-B. B, preassembled TGF-β3·TβRII-His (1:2), mTGF-β3·TβRII-His (1:1), and mmTGF-β2-7M·TβRII-His (1:1) complexes at a concentration of 100 nm (blue bars) or 250 nm (gray bars) were incubated with 50 nm biotinylated TβRI-ΔC-Avi and 2 nm terbium-anti-His and 30 nm SA-665 for 2 h at room temperature. Buffer control (orange bars) contained only 2 nm terbium-anti-His and 30 nm SA-665. Measurements were performed using a BMG Labtech Pherastar FS. ΔF for each sample was determined by assigning two buffer control assays as the negative control as described under “Experimental procedures.”