Alexander Kotzsch

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.

Crystal structure analysis reveals how the Chordin family member crossveinless 2 blocks BMP-2 receptor binding

Crossveinless 2 (CV-2) is an extracellular BMP modulator protein belonging to the Chordin family. During development it is expressed at sites of high BMP signaling and like Chordin CV-2 can either enhance or inhibit BMP activity. CV-2 binds to BMP-2 via its N-terminal Von Willebrand factor type C (VWC) domain 1. Here we report the structure of the complex between CV-2 VWC1 and BMP-2. The tripartite VWC1 binds BMP-2 only through a short N-terminal segment, called clip, and subdomain (SD) 1. Mutational analysis establishes that the clip segment and SD1 together create high-affinity BMP-2 binding. All four receptor-binding sites of BMP-2 are blocked in the complex, demonstrating that VWC1 acts as competitive inhibitor for all receptor types. In vivo experiments reveal that the BMP-enhancing (pro-BMP) activity of CV-2 is independent of BMP-2 binding by VWC1, showing that pro- and anti-BMP activities are structurally separated in CV-2.

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.

Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5-type I receptor specificity.

Dysregulation of growth and differentiation factor 5 (GDF‐5) signalling, a member of the TGF‐β superfamily, is strongly linked to skeletal malformation. GDF‐5‐mediated signal transduction involves both BMP type I receptors, BMPR‐IA and BMPR‐IB. However, mutations in either GDF‐5 or BMPR‐IB lead to similar phenotypes, indicating that in chondrogenesis GDF‐5 signalling seems to be exclusively mediated through BMPR‐IB. Here, we present structural insights into the GDF‐5:BMPR‐IB complex revealing how binding specificity for BMPR‐IB is generated on a molecular level. In BMPR‐IB, a loop within the ligand‐binding epitope functions similar to a latch allowing high‐affinity binding of GDF‐5. In BMPR‐IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF‐5 might impair receptor binding and activation.

Structure analysis of bone morphogenetic protein-2 type I receptor complexes reveals a mechanism of receptor inactivation in juvenile polyposis syndrome.

Bone morphogenetic proteins regulate many developmental processes during embryogenesis as well as tissue homeostasis in the adult. Signaling of bone morphogenetic proteins (BMPs) is accomplished by binding to two types of serine/threonine kinase transmembrane receptors termed type I and type II. Because a large number of ligands signal through a limited number of receptors, ligand-receptor interaction in the BMP superfamily is highly promiscuous, with a ligand binding to various receptors and a receptor binding many different BMP ligands. In this study we investigate the interaction of BMP-2 with its two high affinity type I receptors, BMP receptors IA (BMPR-IA) and BMPR-IB. Interestingly, 50% of the residues in the BMP-2 binding epitope of the BMPR-IA receptor are exchanged in BMPR-IB without a decrease in binding affinity or specificity for BMP-2. Our structural and functional analyses show that promiscuous binding of BMP-2 to both type I receptors is achieved by inherent backbone and side-chain flexibility as well as by variable hydration of the ligand-receptor interface enabling the BMP-2 surface to adapt to different receptor geometries. Despite the high degree of amino acid variability found in BMPR-IA and BMPR-IB binding equally to BMP-2, three single point missense mutations in the ectodomain of BMPR-IA cannot be tolerated. In juvenile polyposis syndrome these mutations have been shown to inactivate BMPR-IA. On the basis of our biochemical and biophysical analyses, we can show that the mutations, which are located outside the ligand binding epitope, alter the local or global fold of the receptor, thereby inactivating BMPR-IA and causing a loss of the BMP-2 tumor suppressor function in colon epithelial cells.

Five Amino Acids in the Innermost Cavity of the Substrate Binding Cleft of Organic Cation Transporter 1 Interact with Extracellular and Intracellular Corticosterone

We have shown previously that Leu447 and Gln448 in the transmembrane helix (TMH) 10 of rat organic cation transporter rOCT1 are critical for inhibition of cation uptake by corticosterone. Here, we tested whether the affinity of corticosterone is different when applied from the extracellular or intracellular side. The affinity of corticosterone was determined by measuring the inhibition of currents induced by tetraethylammonium+ (TEA+) in Xenopus laevis oocytes expressing rOCT1. Either corticosterone and TEA+ were added to the bath simultaneously or the oocytes were preincubated with corticosterone, washed, and TEA+-induced currents were determined subsequently. In mutant L447Y, Ki values for extracellular and intracellular corticosterone were decreased, whereas in mutant Q448E, only the Ki for intracellular corticosterone was changed. Modeling of the interaction of corticosterone with rOCT1 in the inward- or outward-facing conformation predicted direct binding to Leu447, Phe160 (TMH2), Trp218 (TMH4), Arg440 (TMH10), and Asp475 (TM11) from both sides. In mutant F160A, affinities for extracellular and intracellular corticosterone were increased, whereas maximal inhibition was reduced in W218F and R440K. In stably transfected epithelial cells, the affinities for inhibition of 1-methyl-4-phenyl-pyridinium+ (MPP+) uptake by extracellular and intracellular corticosterone were decreased when Asp475 was replaced by glutamate. In mutants F160A, W218Y, R440K, and L447F, the affinities for MPP+ uptake were changed, and in mutant D475E, the affinity for TEA+ uptake was changed. The data suggest that Phe160, Trp218, Arg440, Leu447, and Asp475 are located within an innermost cavity of the binding cleft that is alternatingly exposed to the extracellular or intracellular side during substrate transport.

The solution structure of BMPR-IA reveals a local disorder-to-order transition upon BMP-2 binding.

The structure of the extracellular domain of BMP receptor IA was determined in solution by NMR spectroscopy and compared to its structure when bound to its ligand BMP-2. While most parts of the secondary structure are highly conserved between the bound and unbound forms, large conformational rearrangements can be observed in the beta4beta5 loop of BMPR-IA, which is in contact with BMP-2 and harbors the main binding determinants for the BMPR-IA-BMP-2 interaction. In its unbound form, helix alpha1 in BMPR-IA, which is in the center of the binding epitope for BMP-2, is missing. Since BMP-2 also shows conformational changes in the type I receptor epitope upon binding to BMPR-IA, both binding partners pass through an induced fit mechanism to adapt their binding interfaces to a given interaction surface. The inherent flexibility of both partners possibly explains the promiscuous ligand-receptor interaction observed in the BMP protein superfamily.

Structure Analysis of the IL-5 Ligand-Receptor Complex Reveals a Wrench-like Architecture for IL-5Rα

Interleukin-5 (IL-5) is the key mediator for the function of eosinophil granulocytes, whose deregulation is characteristic of hypereosinophilic diseases and presumably contributes to allergic asthma. IL-5 signaling involves two transmembrane receptors, IL-5Rα and the common β chain, which upon formation of the ternary complex activate the JAK/STAT signaling cascade. To investigate the mechanism underlying ligand-receptor recognition, we determined the structure of IL-5 bound to the extracellular domain of IL-5Rα. IL-5 makes contact with all three fibronectin III-like domains of IL-5Rα, with the receptor architecture resembling a wrench. Mutagenesis data provide evidence that this wrench-like architecture is likely preformed. The structure demonstrates that for steric reasons, homodimeric IL-5 can bind only one receptor molecule, even though two equivalent receptor-binding sites exist. In regard to strong efforts being made to develop IL-5 antagonists for treating asthma and hypereosinophilic diseases, the advances in molecular understanding provided by this structure are of greatest value.

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.

Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens

Chlorination of natural products is often required for their biological activity; notable examples include vancomycin, the last-ditch antibiotic. It is now known that many chlorinated natural products are made not by haloperoxidases, but by FADH2-dependent halogenases. The mechanism of the flavin-containing enzymes is obscure and there are no structural data. Here, crystals of PrnA (tryptophan 7-halogenase), an enzyme that regioselectively chlorinates tryptophan, cocrystallized with tryptophan and FAD are reported. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 67.8, c = 276.9 A. A data set to 1.8 A with 93% completeness and an Rmerge of 7.1% has been collected from a single flash-cooled crystal. A method for incorporating selenomethionine in a Pseudomonas fluorescens expression system also is reported.