Ternary Complex of Transforming Growth Factor-β1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily

Abstract

Transforming growth factor (TGF)-␤1, -␤2, and -␤3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-Å resolution crystal structure of the ternary complex between human TGF-␤1 and the extracellular domains of its type I and type II receptors, T␤RI and T␤RII. The TGF-␤1 ternary complex structure is similar to previously reported TGF-␤3 complex except with a 10°rotation in T␤RI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-␤. T␤RI showed significant binding to TGF-␤2 and TGF-␤3 but not TGF-␤1, and the binding to all three isoforms of TGF-␤ was enhanced considerably in the presence of T␤RII. The preference of TGF-␤2 to T␤RI suggests a variation in its receptor recruitment in vivo. Although TGF-␤1 and TGF-␤3 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-␤ superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of T␤RII on TGF-␤ is unique to TGF-␤s, 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.Transforming growth factor (TGF)-␤ 2 isoforms regulate the growth and differentiation of many cell types involved in normal development, immune function, and carcinogenesis (1-3). TGF-␤s are the founding members of a highly diversified family of signaling ligands and receptors, known as the TGF-␤ superfamily. To date the superfamily consists of more than 30 growth factors and cytokines, including TGF-␤s, bone morphogenetic proteins (BMPs), activins, inhibins, nodal, Müllerian inhibiting substance, growth differentiation factors, and others (4). TGF-␤s and related factors signal through two single-pass transmembrane receptors, known as the type I and type II receptors. These two receptor types have the same overall domain structure, including an extracellular ligand-binding domain displaying a three-finger toxin fold, a single transmembrane helix, and a cytosolic serine-threonine kinase domain. Signaling is initiated by the ligand, which binds the receptor extracellular domains, bringing them together and triggering a phosphorylation cascade, whereby the type II phosphorylates the type I, and the type I phosphorylates Smads, the cytoplasmic effectors of the pathway (3).Specificities have been determined based on cell-based affinity labeling studies with radiolabeled ligands and have enabled th...

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

“…This suggests that mmTGF-␤2-7M functions to inhibit TGF-␤ signaling in the manner anticipated, which is by binding to and blocking endogenous T␤RII. The fact that the measured potency is greater than the greatest affinity previously reported for TGF-␤1 and TGF-␤3 binding to T␤RII (140 nM) (9), suggests that other factors, such as nonspecific association of mmTGF-␤2-7M with the plasma membrane, may serve to potentiate its inhibitory activity.…”

“…The structures of the TGF-␤ receptor complexes (8,9), as well as accompanying binding and cross-linking studies with TGF-␤3 C77S (7,8,37), suggested that the signaling capacity of monomeric TGF-␤s (TGF-␤1 C77S or mTGF-␤1 and TGF-␤3 C77S or mTGF-␤3) arise from their ability to non-covalently dimerize and in turn bind their receptors (Fig. 1, A and C).…”

“…1B) (35,36); second, once formed, these non-covalent dimers would be stabilized as the receptors bind, because crystal structures show that at least one them, T␤RI, binds by straddling the TGF-␤ homodimer interface (Fig. 1, A and C) (8,9).…”

“…The stepwise assembly of T␤RII and T␤RI into a heterotetramer is driven by binding of T␤RI to a composite TGF-␤/T␤RII interface ( Fig. 1A) (8,9).…”

Signaling Receptors for TGF-β Family Members

“…The TGF-b molecule resembles two hand-like structures that are assembled in an antiparallel manner and are held together by a disulfide bond in the "wrist" region. TGF-b contacts TbRI with the "fingertips" and TbRII with the underside of the fingers (Hart et al 2002;Groppe et al 2008;Radaev et al 2010). In addition, direct receptor-receptor interactions contribute to enhanced stability of the receptor-ligand complex (Radaev et al 2010).…”