Ternary complex of transforming growth factor-beta1 reveals isoform-specific ligand recognition and receptor recruitment in the superfamily

Abstract

Transforming growth factor (TGF)-beta1, -beta2, and -beta3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-A resolution crystal structure of the ternary complex between human TGF-beta1 and the extracellular domains of its type I and type II receptors, TbetaRI and TbetaRII. The TGF-beta1 ternary complex structure is similar to previously reported TGF-beta3 complex except with a 10 degrees rotation in TbetaRI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-beta. TbetaRI showed significant binding to TGF-beta2 and TGF-beta3 but not TGF-beta1, and the binding to all three isoforms of TGF-beta was enhanced considerably in the presence of TbetaRII. The preference of TGF-beta2 to TbetaRI suggests a variation in its receptor recruitment in vivo. Although TGF-beta1 and TGF-beta3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-beta superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of TbetaRII on TGF-beta is unique to TGF-betas, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

FIGURE 1.

Ribbon drawing of the TGF-β1 ternary complex. A, top view of the complex. B, side view rotated ∼90° compared with A. TGF-β1 monomers TGF-β1_A and TGF-β1_B are colored cyan and pale cyan, respectively. TβRI and TβRII are colored red and yellow, respectively. C, relative positions of type I receptors from TGF-β1 (red) and TGF-β3 (salmon) ternary complexes. This figure and all subsequent ribbon drawings are prepared using the PyMOL molecular graphics system.

FIGURE 2.

Detailed views of the receptor/ligand interfaces in TGF-β1 ternary complex. A, TβRII/TGF-β1 hydrogen bonding network. B, TβRI/TGF-β1_A interface. C, TβRI/TβRII interface. D, TβRI/TGF-β1_B interface. The central inset is placed as a reference to show location of different interfaces. All of the detailed interfaces are shown as Cα traces. Major residues involved in the interactions are shown in stick representation, colored according to the molecule and labeled. Hydrogen bonds are shown as dotted lines.

FIGURE 3.

Structural superposition between TGF-β1 and TGF-β3 ternary complexes. A and B, interface contacts between TβRI and TGF-β1_A (A) and between TβRI and the helix α1 of TGF-β1_B (B). TGF-β1 complex is colored in light and dark blue for TGF-β1_A and TGF-β1_B monomers and red for TβRI, whereas TGF-β3 complex is in gray. C and D, the TβRII/TGF-β interface (C) and the TβRI/TβRII interface region (D) with TGF-β1 complex colored in light blue for TGF-β1, red for TβRI, and yellow for TβRII, whereas the TGF-β3 complex is in gray.

FIGURE 4.

Structure guided sequence alignment of several ligands and receptors of TGF-β superfamily. A, sequence comparison of several mammalian TGF-βs, BMPs, activin, nodal and growth differentiation factor 5 (GDF5). The numbering is consistent with the sequence of TGF-β1. B, sequence comparison of several type II receptors. The numbering is according to the TβRII sequence. C, sequence alignment of several type I receptors. The numbering is consistent with the TβRI sequence. The secondary structure elements are illustrated as arrows and cylinders for β-strands and α-helices, respectively. The residues involved in interactions in the TGF-β1 and BMP-2 ternary complexes are highlighted cyan and magenta, respectively. Disulfide bonds critical for receptor/ligand specificity and compatibility and highlighted yellow, numbered, and connected by thick yellow lines. Secondary structure elements crucial for receptor/ligand pairing are boxed. Arg²⁵ and Arg⁹⁵ are marked by stars.

FIGURE 5.

Structural determinants critical for receptor/ligand recognition in TGF-β superfamily. A, alignment of TGF-β1 and BMP-2 and their type I receptors. TGF-β1 is colored cyan. For clarity only one monomer of BMP-2 is shown in magenta. TβRI and BMPR-Ia are in red and blue, respectively. β1-β2 and β4-β5 loops as well as disulfide bonds on receptor and ligand that are critical for receptor/ligand compatibility are marked. Receptor II binding sites in TGF-β1 and BMP-2 ternary complex (Site IIa and Site IIb) are outlined as ovals. B, schematic representation of TGF-β-type and BMP-type ternary complex with receptor I and II binding sites marked as Site I, Site IIa, and Site IIb. C, TβRII in surface representation (yellow) with TGF-β1 binding site painted cyan. BMPRII β1-β2 loop is represented as a blue ribbon that blocks the binding site, thus prohibiting BMPRII from binding to Site IIa. D, ActRII in surface representation (gray) with BMP-2 contact area colored magenta. TβRII unique extension of β4-β5 loop (yellow ribbon) and unique conformation of β4-β5 loop in type I receptors (red ribbon) prevent binding of type I receptors and TβRII to Site IIb. E, surface representation of TβRI (red) with TGF-β1 contact area marked cyan. ActRII β1-β2 loop is shown as a gray ribbon that blocks one of the binding sites illustrating impossibility of any type II receptor to bind to Site I. F, critical differences in the conformation between type I (red) and type II (gray) receptors.

FIGURE 6.

Surface plasmon resonance sensorgrams and kinetic fits for binding of the TβRI and TβRII extracellular domains to TGF-β1, -β2, and -β3. A, sensorgrams obtained as TβRII was injected. The traces correspond to triplicate measurements of 2-fold serial dilutions of the receptor over the concentration ranges shown. The surface densities were 185, 339, and 165 RU for TGF-β1, -β2, and -β3, respectively. The red curves correspond to global fits of each data set to a 1:1 binding model using Scrubber 2 software. B, sensorgrams and kinetic fits obtained as TβRI was injected. Surface densities were 242, 339, and 595 RU for TGF-β1, -β2, and -β3, respectively. Sensorgrams obtained for TGF-β3 indicated heterogeneity that could not be fit to a simple 1:1 model, and hence no fit is shown. C, sensorgrams and kinetic fits obtained as TβRI was injected in the presence of 4 μm TβRII. The surface densities were 498, 339, and 595 RU for TGF-β1, -β2, and -β3, respectively.