Neogenin Interacts with Hemojuvelin through Its Two Membrane-Proximal Fibronectin Type III Domains

Hemojuvelin is a recently identified iron-regulatory protein that plays an important role in affecting the expression of hepcidin, a key iron regulatory hormone. Although the underlying mechanism of this process is not clear, several hemojuvelin-binding proteins, including the cell surface receptor neogenin and bone morphogenetic protein (BMP) cytokines, have been identified. The ectodomain of neogenin is composed of four immunoglobulin-like (Ig) domains followed by six fibronectin type III-like (FNIII) domains. Here we report expression of soluble versions of hemojuvelin and neogenin for biochemical characterization of their interaction and the interaction of HJV with BMP-2. Hemojuvelin normally undergoes an autocatalytic cleavage, and as in vivo, recombinant hemojuvelin exists as a mixture of cleaved and uncleaved forms. Neogenin binds to cleaved and noncleaved hemojuvelin, as verified by its binding to an uncleaved mutant hemojuvelin. We localized the hemojuvelin binding site on neogenin to the membrane-proximal fifth and sixth FNIII domains and the juxtamembrane linker and showed that a fragment containing only this region binds 2–3 orders of magnitude more tightly than the entire neogenin ectodomain. Binding to the most membrane-proximal region of neogenin may play a role in regulating the levels of soluble and membrane-bound forms of hemojuvelin, which in turn would influence the amount of free BMP-2 available for binding to its receptors and triggering transcription of the hepcidin gene. Our finding that BMP-2 and neogenin bind simultaneously to hemojuvelin raises the possibility that neogenin is part of a multiprotein complex at the hepatocyte membrane involving BMP, its receptors, and hemojuvelin

Designer TGFβ Superfamily Ligands with Diversified Functionality

Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values

X-ray crystallographic and biochemical studies on the bone morphogenetic protein family

Bone Morphogenetic Proteins (BMPs) are extracellular messenger ligands involved in controlling a wide array of developmental and intercellular signaling processes. To initiate their specific intracellular signaling pathways, the ligands recognize and bind two structurally related serine/threonine kinase receptors, termed type I and type II, on the cell surface. To address the structural arrangement of the receptors when bound to the ligand, the structure of BMP-2, ligand, bound to its type I receptor BMPRIa-ECD and type II receptor ActRII-ECD was determined. The structural arrangement this complete, signaling competent complex confirms that the two receptor types do not directly contact each other. Further, comparison of previously solved high affinity type II receptor/ligand interfaces with the lower affinity interface of BMP-2/ ActRII-ECD allowed for identification of ligand residues important for determining receptor affinity. A known feature of BMP complex assembly is the cooperative nature of receptor binding. When bound to its high affinity receptor, the ligand's affinity for the lower affinity receptor is increased. However, the lack of conformational changes to either the receptors or ligand in the ternary complex leaves the mechanism unclear. Using the natural homo/hetero-dimer system of activin/Inihibin, the nature of this cooperativity was probed. Activin's receptor affinity was shown to vary depending on the surface concentration of the receptor, whereas Inhibin's receptor affinity remained constant. This finding suggests cooperative receptor binding is a result of increased local concentration and loss of rotational freedom of the ligand upon binding to a high affinity receptor. Finally, structural and biochemical studies were undertaken for two new BMP ligands, BMP-3 and BMP-6. Interestingly, while BMP -6 exhibited many similarities to BMP-7, BMP-3 displayed a previously unseen 30-fold specificity difference between ActRIIb-ECD and ActRII-ECD. Comparison of the predicted interfaces of these receptors with BMP-3 yielded a single residue interaction which regulates this receptor preference. The combination of these related studies illustrates how single amino acid differences between ligands can effect receptor binding and, ultimately, impact BMP signaling and functio

X-ray crystallographic and biochemical studies on the bone morphogenetic protein family

Bone Morphogenetic Proteins (BMPs) are extracellular messenger ligands involved in controlling a wide array of developmental and intercellular signaling processes. To initiate their specific intracellular signaling pathways, the ligands recognize and bind two structurally related serine/threonine kinase receptors, termed type I and type II, on the cell surface. To address the structural arrangement of the receptors when bound to the ligand, the structure of BMP-2, ligand, bound to its type I receptor BMPRIa-ECD and type II receptor ActRII-ECD was determined. The structural arrangement this complete, signaling competent complex confirms that the two receptor types do not directly contact each other. Further, comparison of previously solved high affinity type II receptor/ligand interfaces with the lower affinity interface of BMP-2/ ActRII-ECD allowed for identification of ligand residues important for determining receptor affinity. A known feature of BMP complex assembly is the cooperative nature of receptor binding. When bound to its high affinity receptor, the ligand's affinity for the lower affinity receptor is increased. However, the lack of conformational changes to either the receptors or ligand in the ternary complex leaves the mechanism unclear. Using the natural homo/hetero-dimer system of activin/Inihibin, the nature of this cooperativity was probed. Activin's receptor affinity was shown to vary depending on the surface concentration of the receptor, whereas Inhibin's receptor affinity remained constant. This finding suggests cooperative receptor binding is a result of increased local concentration and loss of rotational freedom of the ligand upon binding to a high affinity receptor. Finally, structural and biochemical studies were undertaken for two new BMP ligands, BMP-3 and BMP-6. Interestingly, while BMP -6 exhibited many similarities to BMP-7, BMP-3 displayed a previously unseen 30-fold specificity difference between ActRIIb-ECD and ActRII-ECD. Comparison of the predicted interfaces of these receptors with BMP-3 yielded a single residue interaction which regulates this receptor preference. The combination of these related studies illustrates how single amino acid differences between ligands can effect receptor binding and, ultimately, impact BMP signaling and functio