Walter Sebald

The Mode of Bone Morphogenetic Protein (BMP) Receptor Oligomerization Determines Different BMP-2 Signaling Pathways

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-inducedversus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.

Crystal structure of the BMP-2-BRIA ectodomain complex.

Bone morphogenetic proteins (BMPs) belong to the large transforming growth factor-β (TGF-β) superfamily of multifunctional cytokines. BMP-2 can induce ectopic bone and cartilage formation in adult vertebrates and is involved in central steps in early embryonal development in animals. Signaling by these cytokines requires binding of two types of transmembrane serine/threonine receptor kinase chains classified as type I and type II. Here we report the crystal structure of human dimeric BMP-2 in complex with two high affinity BMP receptor IA extracellular domains (BRIAec). The receptor chains bind to the ‘wrist’ epitopes of the BMP-2 dimer and contact both BMP-2 monomers. No contacts exist between the receptor domains. The model reveals the structural basis for discrimination between type I and type II receptors and the variability of receptor–ligand interactions that is seen in BMP–TGF-β systems.

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.

Crystal Structure of the Interleukin-4/Receptor α Chain Complex Reveals a Mosaic Binding Interface

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.

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.

The proton conducting F0-part of bacterial ATP synthases

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Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement.

Interleukin‐4 (IL‐4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B‐cells and the proliferation of T‐ and B‐cell, and other cell types of the lymphoid system. An antagonist of human IL‐4 was discovered during the studies presented here after Tyr124 of the recombinant protein had been substituted by an aspartic acid residue. This IL‐4 variant, Y124D, bound with high affinity to the IL‐4 receptor (KD = 310 pM), but retained no detectable proliferative activity for T‐cells and inhibited IL‐4‐dependent T‐cell proliferation competitively (K(i) = 620 pM). The loss of efficacy in variant Y124D was estimated to be greater than 100‐fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B‐cells. The substitution of Tyr124 by either phenylalanine, histidine, asparagine, lysine or glycine resulted in partial agonist variants with unaltered receptor binding affinity and relatively small deficiencies in efficacy. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand of a receptor belonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL‐4 receptor system can be derived from the IL‐4 protein.

Structure, binding, and antagonists in the IL-4/IL-13 receptor system.

Interleukin-4 (IL-4) and IL-13 are the only cytokines known to bind to the receptor chain IL-4Ralpha. Receptor sharing by these two cytokines is the molecular basis for their overlapping biological functions. Both are key factors in the development of allergic hypersensitivity, and they also play a major role in exacerbating allergic and asthmatic symptoms. Knowledge of structure and function of this system has allowed the development of inhibitors that block the interaction between the cytokines and their shared receptor. Mutational analysis of IL-4 has revealed variants with high-affinity binding to IL-4Ralpha but no detectable affinity for the second receptor subunit, which is either (gamma)c or IL-13Ralpha1. These IL-4 antagonists fail to induce signal transduction and block IL-4 and IL-13 effects in vitro. IL-4 antagonists prevent the development of allergic disease in vivo and an antagonistic variant of human IL-4 is now in clinical trials for asthma. Detailed knowledge of the site of interaction of IL-4 and IL-4Ralpha has been gained by structure analysis of the complex of these two proteins and through functional studies employing mutants of IL-4 and its receptor subunits. Based on these new data, the hitherto elusive goal of designing small molecular mimetics may be feasible.

Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation.

Interleukin 4 (IL‐4) exerts a decisive role in the coordination of protective immune responses against parasites, particularly helminths. A disregulation of IL‐4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human IL‐4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4‐helix‐bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL‐4 variants deficient in binding to the extracellular domain of the IL‐4 receptor (IL‐4R(ex)). In parallel, up to 1000‐fold increased concentrations of this type of variant were required to induce T‐cell proliferation and B‐cell CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild‐type). IL‐4 variants affected at site 2 exhibited partial agonist activity during T‐cell proliferation; however, they still bound with high affinity to IL‐4R(ex). [The generation of an IL‐4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO J., 11, 3237‐3244)]. These findings indicate that IL‐4 functions by binding IL‐4R(ex) via site 1 which is constituted by residues on helices A and C.(ABSTRACT TRUNCATED AT 250 WORDS)

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.