Bone morphogenetic protein‐2 (BMP‐2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP‐2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor‐β (TGF‐β) superfamily, signals by oligomerizing type I and type II receptor serine‐kinases in the cell membrane. The binding epitopes of BMP‐2 for BMPR‐IA (type I) and BMPR‐II or ActR‐II (type II) were characterized using BMP‐2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high‐affinity BMPR‐IA binding was detected spanning the interface of the BMP‐2 dimer. A smaller epitope 2 for the low‐affinity binding of BMPR‐II was found to be assembled by determinants of a single monomer. Symmetry‐related pairs of the two juxtaposed epitopes occur near the BMP‐2 poles. Mutations in both epitopes yielded variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behaved as antagonists partially or completely inhibiting BMP‐2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF‐β superfamily.

Background: Bone morphogenetic proteins (BMPs) are key regulators in the embryonic development and postnatal tissue homeostasis in all animals. Loss of function or dysregulation of BMPs results in severe diseases or even lethality. Like transforming growth factors β (TGF-βs), activins, growth and differentiation factors (GDFs) and other members of the TGF-β superfamily, BMPs signal by assembling two types of serine/threonine-kinase receptor chains to form a hetero-oligomeric ligand-receptor complex. BMP ligand receptor interaction is highly promiscuous, i.e. BMPs bind more than one receptor of each subtype, and a receptor bind various ligands. The activin type II receptors are of particular interest, since they bind a large number of diverse ligands. In addition they act as high-affinity receptors for activins but are also low-affinity receptors for BMPs. ActR-II and ActR-IIB therefore represent an interesting example how affinity and specificity might be generated in a promiscuous background. Results: Here we present the high-resolution structures of the ternary complexes of wildtype and a variant BMP-2 bound to its high-affinity type I receptor BMPR-IA and its low-affinity type II receptor ActR-IIB and compare them with the known structures of binary and ternary ligand-receptor complexes of BMP-2. In contrast to activin or TGF-β3 no changes in the dimer architecture of the BMP-2 ligand occur upon complex formation. Functional analysis of the ActR-IIB binding epitope shows that hydrophobic interactions dominate in low-affinity binding of BMPs; polar interactions contribute only little to binding affinity. However, a conserved H-bond in the center of the type II ligand-receptor interface, which does not contribute to binding in the BMP-2 – ActR-IIB interaction can be mutationally activated resulting in a BMP-2 variant with high-affinity for ActR-IIB. Further mutagenesis studies were performed to elucidate the binding mechanism allowing us to construct BMP-2 variants with defined type II receptor binding properties. Conclusion: Binding specificity of BMP-2 for its three type II receptors BMPR-II, Act-RII and ActR-IIB is encoded on single amino acid level. Exchange of only one or two residues results in BMP-2 variants with a dramatically altered type II receptor specificity profile, possibly allowing construction of BMP-2 variants that address a single type II receptor. The structure-/function studies presented here revealed a new mechanism, in which the energy contribution of a conserved H-bond is modulated by surrounding intramolecular interactions to achieve a switch between low- and high-affinity binding.

Background: Transforming growth factor (TGF)β superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. Results: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. Conclusion: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.

Bone morphogenetic proteins (BMPs) and other members of the TGF-β superfamily are secreted signalling proteins determining the development, maintenance and regeneration of tissues and organs. These dimeric proteins bind, via multiple epitopes, two types of signalling receptor chains and numerous extracellular modulator proteins that stringently control their activity. Crystal structures of free ligands and of complexes with type I and type II receptor extracellular domains and with the modulator protein Noggin reveal structural epitopes that determine the affinity and specificity of the interactions. Modelling of a ternary complex BMP/(BMPR-IA_EC)₂/(ActR-II_EC)₂suggests a mechanism of receptor activation that does not rely on direct contacts between extracellular domains of the receptors. Mutational and interaction analyses indicate that the large hydrophobic core of the interface of BMP-2 (wrist epitope) with the type I receptor does not provide a hydrophobic hot spot for binding. Instead, main chain amide and carbonyl groups that are completely buried in the contact region represent major binding determinants. The affinity between ligand and receptor chains is probably strongly increased by two-fold interactions of the dimeric ligand and receptor chains that exist as homodimers in the membrane (avidity effects). BMP muteins with disrupted epitopes for receptor chains or modulator proteins provide clues for drug design and development.

Background: Interleukin 4 (IL-4) is a key regulator of the immune system and an important factor in the development of allergic hypersensitivity. Together with interleukin 13 (IL-13), IL-4 plays an important role in exacerbating allergic and asthmatic symptoms. For signal transduction, both cytokines can utilise the same receptor, consisting of the IL-4Rα and the IL-13Rα1 chain, offering an explanation for their overlapping biological functions. Since both cytokine ligands share only moderate similarity on the amino acid sequence level, molecular recognition of the ligands by both receptor subunits is of great interest. IL-4 and IL-13 are interesting targets for allergy and asthma therapies. Knowledge of the binding mechanism will be important for the generation of either IL-4 or IL-13 specific drugs.Results: We present a structure/function analysis of the IL-4 ligand-receptor interaction. Structural determination of a number of IL-4 variants together within vitrobinding studies show that IL-4 and its high-affinity receptor subunit IL-4Rα interact via a modular protein-protein interface consisting of three independently-acting interaction clusters. For high-affinity binding of wild-type IL-4 to its receptor IL-4Rα, only two of these clusters (i.e. cluster 1 centered around Glu9 and cluster 2 around Arg88) contribute significantly to the free binding energy. Mutating residues Thr13 or Phe82 located in cluster 3 to aspartate results in super-agonistic IL-4 variants. All three clusters are fully engaged in these variants, generating a three-fold higher binding affinity for IL-4Rα. Mutagenesis studies reveal that IL-13 utilizes the same main binding determinants,i.e. Glu11 (cluster 1) and Arg64 (cluster 2), suggesting that IL-13 also uses this modular protein interface architecture.Conclusion: The modular architecture of the IL-4-IL-4Rα interface suggests a possible mechanism by which proteins might be able to generate binding affinity and specificity independently. So far, affinity and specificity are often considered to co-vary,i.e. high specificity requires high affinity and vice versa. Although the binding affinities of IL-4 and IL-13 to IL-4Rα differ by a factor of more than 1000, the specificity remains high because the receptor subunit IL-4Rα binds exclusively to IL-4 and IL-13. An interface formed by several interaction clusters/binding hot-spots allows for a broad range of affinities by selecting how many of these interaction clusters will contribute to the overall binding free energy. Understanding how proteins generate affinity and specificity is essential as more and more growth factor receptor families show promiscuous binding to their respective ligands. This limited specificity is, however, not accompanied by low binding affinities.

Background:Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-β (TGF-β) superfamily of multifunctional cytokines. Signaling of the BMPs requires the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR-IB, and