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

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

Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry

AbstractFluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition

A modular interface of IL-4 allows for scalable affinity without affecting specificity for the IL-4 receptor

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 with in vitro binding 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

Anthracycline-Induced Cardiotoxicity: Cardiac Monitoring by Continuous Wave-Doppler Ultrasound Cardiac Output Monitoring and Correlation to Echocardiography

Background: Anthracyclines are agents with a well-known cardiotoxicity. The study sought to evaluate the hemodynamic response to an anthracycline using real-time continuous-wave (CW)-Doppler ultrasound cardiac output monitoring (USCOM) and echocardiography in combination with serum biomarkers. Methods: 50 patients (26 male, 24 female, median age 59 years) suffering from various types of cancer received an anthracycline-based regimen. Patients' responses were measured at different time points (T0 prior to infusion, T1 6 h post infusion, T2 after 1 day, T3 after 7 days, and T4 after 3 months) with CW-Doppler ultrasound (T0-T4) and echocardiography (T1, T4) for hemodynamic parameters such as stroke volume (SV; SVUSCOM ml) and ejection fraction (EF; EFechocardiography%) and with NT-pro-BNP and hs-Troponin T (T0-T4). Results: During the 3-month observation period, the relative decrease in the EF determined by echocardiography was -2.1% (Delta T0-T4, T0 71 +/- 7.8%, T4 69.5 +/- 7%, p = 0.04), whereas the decrease in SV observed using CW-Doppler was -6.5% (Delta T0-T4, T0 54 +/- 19.2 ml, T4 50.5 +/- 20.6 ml, p = 0.14). The kinetics for serum biomarkers were inversely correlated. Conclusions: Combining real-time CW-Doppler USCOM and serum biomarkers is feasible for monitoring the immediate and chronic hemodynamic changes during an anthracycline-based regimen; the results obtained were comparable to those from echocardiography

Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro

Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix (TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix (TM) oligonucleotide array technology. Results were validated by real time RT-PCR. Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye

New Insights into HTLV-1 Particle Structure, Assembly, and Gag-Gag Interactions in Living Cells

Human T-cell leukemia virus type 1 (HTLV-1) has a reputation for being extremely difficult to study in cell culture. The challenges in propagating HTLV-1 has prevented a rigorous analysis of how these viruses replicate in cells, including the detailed steps involved in virus assembly. The details for how retrovirus particle assembly occurs are poorly understood, even for other more tractable retroviral systems. Recent studies on HTLV-1 using state-of-the-art cryo-electron microscopy and fluorescence-based biophysical approaches explored questions related to HTLV-1 particle size, Gag stoichiometry in virions, and Gag-Gag interactions in living cells. These results provided new and exciting insights into fundamental aspects of HTLV-1 particle assembly—which are distinct from those of other retroviruses, including HIV-1. The application of these and other novel biophysical approaches promise to provide exciting new insights into HTLV-1 replication

Receptor oligomerization and beyond: a case study in bone morphogenetic proteins

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

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

<p>Abstract</p> <p>Background</p> <p>Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an <it>EYFP </it>reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells.</p> <p>Results</p> <p>The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions.</p> <p>Conclusions</p> <p>In summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry.</p