Transforming growth factor β isoforms (TGF-β) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-β play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-β type I receptor (TβR-I) and TGF-β type II receptor (TβR-II). TGF-β's specificity for TβR-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-β and TβR-II. The structure and backbone dynamics of the unbound form of the TβR-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the β-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 310 helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-β signaling complex with its unique specificity, departing from the ancestral promiscuity of the bone morphogenetic protein subfamily, where the binding interface of the type I receptor is highly flexible.

Bone morphogenetic proteins (BMPs) are secreted signaling proteins – they transduce their signals by by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homolog Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the β-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the β4-β5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the β4-β5 loop, and to lesser extent β1-β2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9:Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3, but is rotated by 40 degrees. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the β4-β5 loop that contribute significantly to binding and are non-conservatively substituted relative to the corresponding residues in Alk3.

Edited by Norma M. Allewell Betaglycan (BG) is a membrane-bound co-receptor of the TGF-␤ family that selectively binds transforming growth factor-␤ (TGF-␤) 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-␤2 with BG's C-terminal binding domain, BG ZP-C , and surface plasmon resonance binding measurements with TGF-␤2 variants, we found that the BG ZP-C -binding site on TGF-␤2 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-␤ isoforms and the InhA ␣-subunit, but they are unconserved in other TGF-␤ 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-␤2 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-␤2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-␤2 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-␤2 and shed light on the specificity of BG for select TGF-␤-type GFs and the mechanisms by which BG influences their signaling.Betaglycan (BG) 3 is a co-receptor of the highly diversified transforming growth factor ␤ family (TGF-␤) of signaling proteins, which have essential roles in diverse processes, ranging from patterning of embryos and organs in embryogenesis to regulation of the endocrine and adaptive immune systems in adults (1-3). BG is expressed by epithelial, mesenchymal, and other cell types, but unlike the type I and type II signaling receptors (4 -6) that bind TGF-␤ family growth factors (GFs) to transduce the signal by phosphorylating Smads and other effectors (7-9), it is not required for signal transduction (10). BG is nonetheless essential in vertebrates, such as mice, where knockout of the BG gene is embryonic lethal (11).BG is a membrane-anchored proteoglycan composed of a large extracellular domain (766 amino acids in humans), a single transmembrane-spanning helix, and a short (43 amino acids in humans) highly-conserved cytoplasmic tail (Fig. 1A). BG's extracellular domain is composed of two subdomains, each roughly equal in size: the membrane-distal orphan domain (BG O ) and the C-terminal membrane-proximal zona pellucida domain (BG ZP ). The structure of BG's orphan domain is not known, but it is likely two tandem ␤-sandwiches, designated BG OR1 and BG OR2 , similar to that of endoglin (12), a homologous co-receptor of the TGF-␤ family with the same overall domain structure (13). BG ZP is likely composed of tandem immunoglobulin-like domains, d...

Beta2-glycoprotein I (β2GPI) is the most common antigen for autoimmune antibodies in antiphospholipid syndrome (APS). Thrombosis is a clinical feature of APS. We made a molecule (A1-A1) that consists of two identical β2GPI-binding modules from ApoE receptor 2. A1-A1 binds to β2GPI/anti-β2GPI antibody complexes preventing their association with ApoER2 and anionic phospholipids, and reducing thrombus size in the mouse model of APS. Here, we describe a mutant of A1-A1 (mA1-A1ND) with improved affinity for β2GPI. mA1-A1ND inhibits the binding of β2GPI to cardiolipin in the presence of anti-β2GPI antibodies and inhibits β2GPI/antibody complexes in plasma samples of APS patients affecting the clotting time. Inhibition of the clotting time demonstrates the presence of soluble β2GPI/anti-β2GPI antibody complexes in patients’ plasma. These complexes either already exist in patients’ plasma or form rapidly in the vicinity of phospholipids. All members of the LDL receptor family can bind β2GPI. Modelling studies of A1 in a complex with domain V of β2GPI (β2GPI-DV) revealed two possible orientations of a ligand-binding module from lipoprotein receptors on β2GPI-DV. In both orientations, the ligand-binding module interferes with the binding of β2GPI to anionic phospholipids, however it interacts with two different though overlapping sets of lysine residues in β2GPI-DV, depending on the orientation.