Joachim Nickel

BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II

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.

Molecular recognition of BMP-2 and BMP receptor IA

Bone morphogenetic protein-2 (BMP-2) and other members of the TGF-β superfamily regulate the development, maintenance and regeneration of tissues and organs. Binding epitopes for these extracellular signaling proteins have been defined, but hot spots specifying binding affinity and specificity have so far not been identified. In this study, mutational and structural analyses show that epitopes of BMP-2 and the BRIA receptor form a new type of protein-protein interface. The main chain atoms of Leu 51 and Asp53 of BMP-2 represent a hot spot of binding to BRIA. The BMP-2 variant L51P was deficient in type I receptor binding only, whereas its overall structure and its binding to type II receptors and modulator proteins, such as noggin, were unchanged. Thus, the L51P substitution converts BMP-2 into a receptor-inactive inhibitor of noggin. These results are relevant for other proteins of the TGF-β superfamily and provide useful clues for structure-based drug design.

Promiscuity and specificity in BMP receptor activation

Bone Morphogenetic Proteins (BMPs), together with Transforming Growth Factor (TGF)‐β and Activins/Inhibins constitute the TGF‐β superfamily of ligands. This superfamily is formed by more than 30 structurally related secreted proteins. Since TGF‐β members act as morphogens, either a strict relation between a particular ligand to a distinct cellular receptor and/or temporospatial expression patterns of ligands and receptors is expected. Instead, only a limited number of receptors exist implicating promiscuous interactions of ligands and receptors. Furthermore, in complex tissues a multitude of different ligands can be found, which signal via overlapping subsets of receptors. This raises the intriguing question how concerted interactions of different ligands and receptors generate highly specific cellular signals, which are required during development and tissue homeostasis.

CCM3 interacts with CCM2 indicating common pathogenesis for cerebral cavernous malformations.

Individuals carrying a mutation in one of the three cerebral cavernous malformation genes (CCM1/KRIT1, CCM2, CCM3) cannot be clinically distinguished, raising the possibility that they act within common molecular pathways. In this study, we demonstrate that CCM3 (PDCD10) coprecipitates and colocalizes with CCM2. We also show that CCM3 directly binds to serine/threonine kinase 25 (STK25, YSK1, SOK1) and the phosphatase domain of Fas-associated phosphatase-1 (FAP-1, PTPN13, PTP-Bas, PTP-BL). CCM3 is phosphorylated by STK25 but not by its other Yeast-Two hybrid interactor STK24, whereas the C-terminal catalytic domain of FAP-1 dephosphorylates CCM3. Finally, our experiments reveal that STK25 forms a protein complex with CCM2. Thus, our data link two proteins of unknown function, CCM3 and STK25, with CCM2, which is part of signaling pathways essential for vascular development and CCM pathogenesis.

Molecular recognition in bone morphogenetic protein (BMP)/receptor interaction

Abstract 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-IAEC)2/(ActR-IIEC)2 suggests 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.

A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor

BackgroundBone 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.ResultsHere 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.ConclusionBinding 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.

von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

Bone morphogenetic protein (BMP) function is regulated in the extracellular space by many modulator proteins, including those containing a von Willebrand factor type C (VWC) domain. The function of the VWC domain-containing proteins in development and diseases has been extensively studied. The structural basis, however, for the mechanism by which BMP is regulated by these proteins is still poorly understood. By analyzing chordin, CHL2 (chordin-like 2), and CV2 (crossveinless 2) as well as their individual VWC domains, we show that the VWC domain is a versatile binding module that in its multiple forms and environments can expose a variety of binding specificities. Three of four, two of three, and one of five VWCs from chordin, CHL2, and CV2, respectively, can bind BMPs. Using an array of BMP-2 mutant proteins, it can be demonstrated that the binding-competent VWC domains all use a specific subset of BMP-2 binding determinants that overlap with the binding site for the type II receptors (knuckle epitope) or for the type I receptors (wrist epitope). This explains the competition between modulator proteins and receptors for BMP binding and therefore the inhibition of BMP signaling. A subset of VWC domains from CHL2 binds to the Tsg (twisted gastrulation) protein similar to chordin. A stable ternary complex consisting of BMP-2, CHL2, and Tsg can be formed, thus making CHL2 a more efficient BMP-2 inhibitor. The VWCs of CV2, however, do not interact with Tsg. The present results show that chordin, CHL2, and CV2 regulate BMP-2 signaling by different recognition mechanisms.

Intricacies of BMP receptor assembly

The TGF-beta superfamily exhibits a feature making it distinct from many other growth factor families in that the inadequate number of ligands and receptors premises a high degree of promiscuity in ligand-receptor interaction. This highlights the importance of even small differences in affinities and specificities between different binding partners to maintain the broad spectrum of their well defined biological functions. Despite the promiscuous interactions recent data reveal differences in receptor recruitment, architectures of these assemblies and specific modulation by a multitude of extracellular as well as membrane-associated factors. These modulatory mechanisms might possibly add specificity towards defined biological functions despite the overlapping usage of receptors by various ligands.

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

Type I receptor binding of bone morphogenetic protein 6 is dependent on N‐glycosylation of the ligand

Bone morphogenetic proteins (BMPs), together with transforming growth factor (TGF)‐β and activins/inhibins, constitute the TGF‐β superfamily of ligands. This superfamily is formed by more than 30 structurally related secreted proteins. The crystal structure of human BMP‐6 was determined to a resolution of 2.1 Å; the overall structure is similar to that of other TGF‐β superfamily ligands, e.g. BMP‐7. The asymmetric unit contains the full dimeric BMP‐6, indicating possible asymmetry between the two monomeric subunits. Indeed, the conformation of several loops differs between both monomers. In particular, the prehelix loop, which plays a crucial role in the type I receptor interactions of BMP‐2, adopts two rather different conformations in BMP‐6, indicating possible dynamic flexibility of the prehelix loop in its unbound conformation. Flexibility of this loop segment has been discussed as an important feature required for promiscuous binding of different type I receptors to BMPs. Further studies investigating the interaction of BMP‐6 with different ectodomains of type I receptors revealed that N‐glycosylation at Asn73 of BMP‐6 in the wrist epitope is crucial for recognition by the activin receptor type I. In the absence of the carbohydrate moiety, activin receptor type I‐mediated signaling of BMP‐6 is totally diminished. Thus, flexibility within the binding epitope of BMP‐6 and an unusual recognition motif, i.e. an N‐glycosylation motif, possibly play an important role in type I receptor specificity of BMP‐6.